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Publication number	US20050186573 A1
Publication type	Application
Application number	US 10/625,755
Publication date	Aug 25, 2005
Filing date	Jul 24, 2003
Priority date	Jul 24, 2002
Publication number	10625755, 625755, US 2005/0186573 A1, US 2005/186573 A1, US 20050186573 A1, US 20050186573A1, US 2005186573 A1, US 2005186573A1, US-A1-20050186573, US-A1-2005186573, US2005/0186573A1, US2005/186573A1, US20050186573 A1, US20050186573A1, US2005186573 A1, US2005186573A1
Inventors	Richard Janeczko
Original Assignee	Janeczko Richard A.
Export Citation	BiBTeX, EndNote, RefMan
Patent Citations (8), Referenced by (5), Classifications (11), Legal Events (2)

External Links: USPTO, USPTO Assignment, Espacenet

Polynucleotides for use as tags and tag complements in the detection of nucleic acid sequences

US 20050186573 A1

Abstract

A family of minimally cross-hybridizing nucleotide sequences and their use in the detection of nucleic acid sequences is described. Specifically described is the use of two distinct families of 210 and 1168 24mers in the detection of nucleic acid sequences.

Images(7)

Claims(69)

1. A composition comprising a cleavage structure, said cleavage structure comprising:

a) a target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region and said second region is located to and downstream from said third region;

b) a first oligonucleotide having a 5′ portion and a 3′ portion, said 5′ portion of said first oligonucleotide having a sequence complementary to said second region of said target nucleic acid and said 3′ portion of said first oligonucleotide having a sequence complementary to said third region of said target nucleic acid; and

c) a second oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central region of said second oligonucleotide having a sequence complementary to said second region of said target nucleic acid, and said 3′ portion of said second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides based on a following group of sequences,

1 4 6 6 1 3 2 4 5 5 2 3 1 8 1 2 3 4 1 7 1 9 8 4 1 1 9 2 6 9 1 2 4 3 9 6 9 8 9 8 10 9 9 1 2 3 8 10 8 8 7 4 3 1 1 1 1 1 1 2 2 1 3 3 2 2 3 1 2 2 3 2 4 1 4 4 4 2 1 2 3 3 1 1 1 3 2 2 1 4 3 3 3 3 3 4 4 3 1 1 4 4 3 4 1 1 3 3 3 6 6 6 3 5 6 6 1 1 6 5 7 6 7 7 7 5 8 7 5 5 8 8 2 1 7 7 1 1 2 3 2 3 1 3 2 6 5 6 1 6 4 8 1 1 3 8 5 3 1 1 6 3 5 6 8 8 6 6 8 3 6 5 7 3 1 2 3 1 4 6 1 5 7 5 4 3 2 1 6 7 3 6 2 6 1 3 3 1 2 7 6 8 3 1 3 4 3 1 2 5 3 5 6 1 2 7 3 6 1 7 2 7 4 6 3 5 1 7 5 4 6 3 8 6 6 8 2 3 7 1 7 1 7 8 6 3 7 3 4 1 6 8 4 7 7 1 2 4 3 6 5 2 6 3 1 4 1 4 6 1 3 3 1 4 8 1 8 3 3 5 3 8 1 3 6 6 3 7 7 3 8 6 4 7 3 1 3 7 8 6 10 9 5 5 10 10 7 10 10 10 7 9 9 9 7 7 10 9 9 3 10 3 10 3 9 6 3 4 10 6 10 4 10 3 9 4 3 9 3 10 4 9 9 10 5 9 4 8 3 9 4 9 10 7 3 5 9 4 10 8 4 10 5 4 9 3 5 3 3 9 8 10 6 8 6 9 7 10 4 6 10 9 6 4 4 9 8 10 8 3 7 7 9 10 5 3 8 8 9 3 9 10 8 10 2 9 5 9 9 6 2 2 7 10 9 7 5 3 10 6 10 3 6 8 9 2 10 9 3 2 7 3 8 9 10 3 6 2 3 2 5 10 8 9 8 2 3 10 2 9 6 3 9 8 2 10 3 7 3 9 9 10 9 10 1 1 9 4 10 1 9 1 4 1 7 1 10 9 8 1 9 1 10 1 10 6 9 6 9 1 3 10 3 10 8 8 9 1 3 8 1 9 10 3 9 10 1 3 6 9 1 9 1 10 3 1 1 4 9 6 8 10 3 3 9 6 1 10 5 3 1 6 9 10 6 1 8 10 9 6 5 9 9 4 10 3 2 10 9 1 9 5 10 10 7 2 1 9 10 9 9 1 8 2 1 8 6 8 9 10 1 9 1 3 8 10 9 6 9 10 1 2 1 10 8 9 9 2 1 9 6 7 2 9 4 3 9 3 5 1 5 11 10 14 12 1 7 12 4 13 3 2 5 5 4 4 12 9 2 13 13 11 13 13 10 2 5 4 12 7 11 7 4 11 6 4 12 12 1 9 11 11 12 9 4 14 12 6 12 7 13 2 9 11 9 11 3 4 1 3 10 5 12 11 4 4 4 13 7 12 1 5 9 13 10 11 11 6 10 14 14 10 1 3 2 14 1 10 4 5 10 12 12 7 11 10 9 11 2 12 8 11 2 8 5 2 12 14 1 8 13 3 7 8 9 4 7 5 4 2 13 2 12 7 1 12 11 10 9 7 5 11 8 12 2 2 12 7 5 2 14 3 4 13 1 8 8 1 5 9 14 5 11 10 13 3 14 1 4 13 2 4 4 4 5 11 3 10 10 9 2 3 3 11 11 4 8 14 3 4 5 1 14 8 11 2 14 3 11 6 12 5 13 4 4 1 10 1 6 10 11 6 5 1 5 8 12 5 1 7 4 5 9 6 9 2 13 2 4 4 2 3 11 2 2 5 9 3 8 1 10 12 2 8 12 7 9 11 4 1 12 1 4 14 3 13 11 2 7 10 4 1 3 4 12 11 11 11 3 3 4 2 12 11 1 5 9 4 2 1 6 1 12 2 10 5 10 5 1 12 2 14 2 11 7 9 4 11 7 4 4 5 14 12 12 5 2 1 10 12 5 9 2 11 6 1 12 14 3 6 1 14 5 9 11 10 1 4 2 5 12 14 10 10 4 5 8 4 5 6 10 12 4 6 12 5 4 2 1 13 6 8 9 10 10 14 5 3 6 14 10 11 3 3 2 9 10 12 5 7 13 3 7 10 5 12 6 4 1 2 5 13 6 1 13 4 14 13 2 12 1 14 1 9 4 11 13 2 6 10 1 10 7 4 5 8 7 2 2 10 13 4 8 2 11 4 6 14 4 8 2 6 2 3 7 1 12 11 2 9 5 6 10 4 13 4 5 10 4 11 9 3 3 11 9 3 2 3 8 15 6 20 17 19 21 10 15 3 7 11 11 7 17 20 14 9 16 6 17 13 21 21 10 15 22 6 17 21 15 7 17 10 22 22 3 20 8 15 20 16 17 21 10 16 6 22 6 21 14 14 14 16 7 17 3 20 10 7 16 19 14 17 7 21 20 16 7 15 22 10 20 10 18 11 22 18 18 7 19 15 7 22 21 18 7 21 16 3 14 13 7 22 17 13 19 7 8 12 10 17 15 3 21 14 9 7 19 6 15 7 14 14 4 17 10 15 20 19 21 6 18 4 20 16 2 19 8 17 6 13 12 12 6 17 4 20 16 21 12 10 19 16 14 14 15 2 7 21 8 16 21 6 22 16 14 17 22 14 17 20 10 21 7 15 21 18 16 13 20 18 21 12 15 7 4 22 14 13 7 19 14 8 15 4 4 5 3 20 7 16 22 18 6 18 13 20 19 6 16 3 13 3 18 6 22 7 20 18 10 17 11 21 8 13 7 10 17 19 10 14

wherein:

(A) each of 1 to 22 is a 4mer selected from the group of 4mers consisting of WWWW, WWWX, WWWY, WWXW, WWXX, WWXY, WWYW, WWYX, WWYY, WXWW, WXWX, WXWY, WXXW, WXXX, WXXY, WXYW, WXYX, WXYY, WYWW, WYWX, WYWY, WYXW, WYXX, WYXY, WYYW, WYYX, WYYY, XWWW, XWWX, XWWY, XWXW, XWXX, XWXY, XWYW, XWYX, XWYY, XXWW, XXWX, XXWY, XXXW, XXXX, XXXY, XXYW, XXYX, XXYY, XYWW, XYWX, XYWY, XYXW, XYXX, XYXY, XYYW, XYYX, XYYY, YWWW, YWWX, YWWY, YWXW, YWXX, YWXY, YWYW, YWYX, YWYY, YXWW, YXWX, YXWY, YXXW, YXXX, YXXY, YXYW, YXYX, YXYY, YYWW, YYWX, YYWY, YYXW, YYXX, YYXY, YYYW, YYYX, and YYYY, and

(B) each of 1 to 22 is selected so as to be different from all of the others of 1 to 22;

(i) (a) W=one of A, T/U, G, and C,

X=one of A, T/U, G, and C,

Y=one of A, T/U, G, and C,

and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,

(b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y, or

(ii) (a) W=G or C,

X=A or T/U,

Y=A or T/U,

and X≠Y, and

(b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in all the sequences;

(D) up to three bases can be inserted at any location of any of the sequences or up to three bases can be deleted from any of the sequences;

(E) all of the sequences of a said group of oligonucleotides are read 5′ to 3′ or are read 3′ to 5′; and

wherein each oligonucleotide of a said set has a sequence of at least ten contiguous bases of the sequence on which it is based, provided that:

(F) (I) the quotient of the sum of G and C divided by the sum of A, T/U, G and C for all combined sequences of the set is between about 0.1 and 0.40 and said quotient for each sequence of the set does not vary from the quotient for the combined sequences by more than 0.2; and

(II) for any phantom sequence generated from any pair of first and second sequences of the set L₁and L₂in length, respectively, by selection from the first and second sequences of identical bases in identical sequence with each other:

(i) any consecutive sequence of bases in the phantom sequence which is identical to a consecutive sequence of bases in each of the first and second sequences from which it is generated is less than ((¾×L)−1) bases in length;

(ii) the phantom sequence, if greater than or equal to (⅚×L) in length, contains at least three insertions/deletions or mismatches when compared to the first and second sequences from which itis generated; and

(iii) the phantom sequence is not greater than or equal to ( 11/12×L) in length;

where L=L₁, or if L₁≠L₂, where L is the greater of L₁and L₂; and

wherein any base present may be substituted by an analogue thereof.

2. The composition of claim 1, wherein one or more of said first and second oligonucleotides contain a 3′-terminal dideoxynucleotide.

3. The composition of claim 1, wherein the composition includes a plurality of said target nucleic acid sequences and a plurality of second oligonucleotide molecules such that each of said second oligonucleotide molecules has a distinct 3′ region.

4. The composition of claim 3, wherein the composition includes at least ten said second oligonucleotide molecules or at least eleven said second oligonucleotide molecules, or at least twelve said second oligonucleotide molecules, or at least thirteen said second oligonucleotide molecules, or at least fourteen said second oligonucleotide molecules, or at least fifteen said second oligonucleotide molecules, or at least sixteen said second oligonucleotide molecules, or at least seventeen said second oligonucleotide molecules, or at least eighteen said second oligonucleotide molecules, or at least nineteen said second oligonucleotide molecules, or at least twenty said second oligonucleotide molecules, or at least twenty-one said second oligonucleotide molecules, or at least twenty-two said second oligonucleotide molecules, or at least twenty-three said second oligonucleotide molecules, or at least twenty-four said second oligonucleotide molecules, or at least twenty-five said second oligonucleotide molecules, or at least twenty-six said second oligonucleotide molecules, or at least twenty-seven said second oligonucleotide molecules, or at least twenty-eight said second oligonucleotide molecules, or at least twenty-nine said second oligonucleotide molecules, or at least thirty said second oligonucleotide molecules, or at least thirty-one said second oligonucleotide molecules, or at least thirty-two said second oligonucleotide molecules, or at least thirty-three said second oligonucleotide molecules, or at least thirty-four said second oligonucleotide molecules, or at least thirty-five said second oligonucleotide molecules, or at least thirty-six said second oligonucleotide molecules, or at least thirty-seven said second oligonucleotide molecules, or at least thirty-eight said second oligonucleotide molecules, or at least thirty-nine said second oligonucleotide molecules, or at least forty said second oligonucleotide molecules, or at least forty-one said second oligonucleotide molecules, or at least forty-two said second oligonucleotide molecules, or at least forty-three said second oligonucleotide molecules, or at least forty-four said second oligonucleotide molecules, or at least forty-five said second oligonucleotide molecules, or at least forty-six said second oligonucleotide molecules, or at least forty seven said second oligonucleotide molecules, or at least forty-eight said second oligonucleotide molecules, or at least forty-nine said second oligonucleotide molecules, or at least fifty said second oligonucleotide molecules, or at least sixty said second oligonucleotide molecules, or at least seventy said second oligonucleotide molecules, or at least eighty said second oligonucleotide molecules, or at least ninety said second oligonucleotide molecules, or at least one hundred said second oligonucleotide molecules, or at least one hundred and ten said second oligonucleotide molecules, or at least one hundred and twenty said second oligonucleotide molecules, or at least one hundred and thirty said second oligonucleotide molecules, or at least one hundred and forty said second oligonucleotide molecules, or at least one hundred and fifty said second oligonucleotide molecules, or at least one hundred and sixty said second oligonucleotide molecules, or at least one hundred and seventy said second oligonucleotide molecules, or at least one hundred and eighty said second oligonucleotide molecules, or at least one hundred and ninety said second oligonucleotide molecules, or at least two hundred said second oligonucleotide molecules.

5. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products, the method comprising:

a) providing:

i) a cleavage means,

ii) a target nucleic acid, said target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region;

iii) a first oligonucleotide having a 5′ and a 3′ portion, said 5′ portion of said first oligonucleotide having a sequence complementary to said second region of said target nucleic acid and said 3′ portion of said first oligonucleotide having a sequence complementary to said third region of said target nucleic acid;

iv) a second oligonucleotide having a 5, portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central portion of said second oligonucleotide having a sequence complimentary to said second region of said target nucleic acid, and said 3′ portion of said second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides based one a following group of sequences;

wherein:

(B) each of 1 to 22 is selected so as to be different from all of the others of 1 to 22;

(i) (a) W=one of A, T/U, G, and C,

X=one of A, T/U, G, and C,

Y=one of A, T/U, G, and C,

and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,

(b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y, or

(ii) (a) W=G or C,

X=A or T/U,

Y A or T/U,

and X≠Y, and

(b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in all the sequences;

(D) up to three bases can be inserted at any location of any of the sequences or up to three bases can be deleted from any o f the sequences;

(E) all of the sequences of a said group of oligonucleotides are read 5′ to 31 or are read 3′ to 5′; and

wherein each oligonucleotide of a said set has a sequence of at least ten contiguous bases of the sequence on which it is based, provided that:

(iii) the phantom sequence is not greater than or equal to ( 11/12×L) in length;

where L=L₁, or if L₁≠L₂, where L is the greater of L₁and L2; and

wherein any base present may be substituted by an analogue thereof;

b) mixing said cleavage means, said target nucleic acid, said first and second oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products; and

c) detecting said non-target cleavage products.

6. The method of claim 5, wherein said reaction temperature is between approximately 50 and 70 degrees centigrade.

7. The method of claim 5, wherein said target nucleic acid comprises single-stranded DNA.

8. The method of claim 5, wherein said target nucleic acid comprises double-stranded DNA and prior to step (c), said reaction mixture is treated such that said double-stranded DNA is rendered substantially single-stranded.

9. The method of claim 5, wherein said treatment to render said double-stranded DNA is rendered substantially single-stranded by increasing the temperature.

10. The method of claim 5, wherein said target nucleic acid comprises RNA and wherein said first and second oligonucleotides comprise DNA.

11. The method of claim 5, wherein said cleavage means comprises a thermostable 5′ nuclease.

12. The method of claim 11, wherein a portion of the amino acid sequence is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a thermophilic organism.

13. The method of claim 12, wherein said organism is selected from the group consisting of Thermus aquaticus, Thermus flavus and Thermus thermophilus.

14. The method of claim 5, wherein said source of target nucleic acid comprises a sample containing genomic DNA.

15. The method of claim 5, wherein said reaction conditions comprise providing a source of divalent cations.

16. The method of claim 15, wherein said divalent cation is selected from the group comprising Mn²⁺ and Mg²⁺ ions.

17. The method of claim 5, wherein the method includes a plurality of said target nucleic acid sequences and a plurality of said second oligonucleotide molecules such that each of said second oligonucleotide molecules has a distinct 3′ region.

18. The method of claim 5, wherein the method includes at least ten said second oligonucleotide molecules or at least eleven said second oligonucleotide molecules, or at least twelve said second oligonucleotide molecules, or at least thirteen said second oligonucleotide molecules, or at least fourteen said second oligonucleotide molecules, or at least fifteen said second oligonucleotide molecules, or at least sixteen said second oligonucleotide molecules, or at least seventeen said second oligonucleotide molecules, or at least eighteen said second oligonucleotide molecules, or at least nineteen said second oligonucleotide molecules, or at least twenty said second oligonucleotide molecules, or at least twenty-one said second oligonucleotide molecules, or at least twenty-two said second oligonucleotide molecules, or at least twenty-three said second oligonucleotide molecules, or at least twenty-four said second oligonucleotide molecules, or at least twenty-five said second oligonucleotide molecules, or at least twenty-six said second oligonucleotide molecules, or at least twenty-seven said second oligonucleotide molecules, or at least twenty-eight said second oligonucleotide molecules, or at least twenty-nine said second oligonucleotide molecules, or at least thirty said second oligonucleotide molecules, or at least thirty-one said second oligonucleotide molecules, or at least thirty-two said second oligonucleotide molecules, or at least thirty-three said second oligonucleotide molecules, or at least thirty-four said second oligonucleotide molecules, or at least thirty-five said second oligonucleotide molecules, or at least thirty-six said second oligonucleotide molecules, or at least thirty-seven said second oligonucleotide molecules, or at least thirty-eight said second oligonucleotide molecules, or at least thirty-nine said second oligonucleotide molecules, or at least forty said second oligonucleotide molecules, or at least forty-one said second oligonucleotide molecules, or at least forty-two said second oligonucleotide molecules, or at least forty-three said second oligonucleotide molecules, or at least forty-four said second oligonucleotide molecules, or at least forty-five said second oligonucleotide molecules or at least forty-six said second oligonucleotide molecules, or at least forty-seven said second oligonucleotide molecules, or at least forty-eight said second oligonucleotide molecules, or at least forty-nine said second oligonucleotide molecules, or at least fifty said second oligonucleotide molecules, or at least sixty said second oligonucleotide molecules, or at least seventy said second oligonucleotide molecules, or at least eighty said second oligonucleotide molecules, or at least ninety said second oligonucleotide molecules, or at least one hundred said second oligonucleotide molecules, or at least one hundred and ten said second oligonucleotide molecules, or at least one hundred and twenty said second oligonucleotide molecules, or at least one hundred and thirty said second oligonucleotide molecules, or at least one hundred and forty said second oligonucleotide molecules, or at least one hundred and fifty said second oligonucleotide molecules, or at least one hundred and sixty said second oligonucleotide molecules, or at least one hundred and seventy said second oligonucleotide molecules, or at least one hundred and eighty said second oligonucleotide molecules, or at least one hundred and ninety said second oligonucleotide molecules, or at least two hundred said second oligonucleotide molecules.

19. The method of claim 5, wherein said 3′ portion of said second oligonucleotide incorporates fluorescent molecule, a radiolabelled nucleotide, digoxigenin, biotinylation and the like.

20. A method of analyzing a biological sample comprising a plurality of target nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule, the method comprising:

a) providing:

i) a cleavage means,

ii) a plurality of target nucleic acid molecules, each of said target nucleic acid molecules having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region;

iii) a plurality of first oligonucleotide molecules, each having a 5′ and a 3′ portion, said 5′ portion of said first oligonucleotide molecules having a sequence complementary to said second region of said target nucleic acid molecules and said 3′ portion of said first oligonucleotide molecules having a sequence complementary to said third region of said target nucleic acid molecules;

iv) a plurality of second oligonucleotide molecules, each having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide molecules having a sequence complementary to said first region of said target nucleic acid molecules, said central portion of said second oligonucleotide molecules having a sequence complimentary to said second region of said target nucleic acid molecules, and said 3′ portion of said second oligonucleotide molecules having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides based on a following group of sequences,

wherein:

(B) each of 1 to 22 is selected so as to be different from all of the others of 1 to 22;

(i) (a) W=one of A, T/U, G, and C,

X=one of A, T/U, G, and C,

Y=one of A, T/U, G, and C,

and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,

(b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y, or

(ii) (a) W=G or C,

X=A or T/U,

Y=A or T/U,

and X≠Y, and

(b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in all the sequences;

(D) up to three bases can be inserted at any location of any of the sequences or up to three bases can be deleted from any of the sequences;

(E) all of the sequences of a said group of oligonucleotides are read 5′ to 3′ or are read 3′ to 5′; and

wherein each oligonucleotide of a said set has a sequence of at least ten contiguous bases of the sequence on which it is based, provided that:

(iii) the phantom sequence is not greater than or equal to ( 11/12×L) in length;

where L=L₁, or if L₁≠L₂, where L is the greater of L₁and L₂; and

wherein any base present may be substituted by an analogue thereof;

c) detecting said non-target cleavage products.

21. The method of claim 20, wherein said reaction temperature is between approximately 50 and 70 degrees centigrade.

22. The method of claim 20, wherein said target nucleic acid molecules comprises single-stranded DNA.

23. The method of claim 20, wherein said target nucleic acid molecules comprises double-stranded DNA and prior to step (c), said reaction mixture is treated such that said double-stranded DNA is rendered substantially single-stranded.

24. The method of claim 20, wherein said treatment to render said double-stranded DNA is rendered substantially single-stranded by increasing the temperature.

25. The method of claim 20, wherein said target nucleic acid molecules comprises RNA and wherein said first and second oligonucleotide molecules comprise DNA.

26. The method of claim 20, wherein said cleavage means comprises a thermostable 5′ nuclease.

27. The method of claim 26, wherein a portion of the amino acid sequence is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a thermophilic organism.

28. The method of claim 28, wherein said organism is selected from the group consisting of Thermus aquaticus, Thermus flavus and Thermus thermophilus.

29. The method of claim 20, wherein said source of target nucleic acid molecules comprises a sample containing genomic DNA.

30. The method of claim 20, wherein said reaction conditions comprise providing a source of divalent cations.

31. The method of claim 30, wherein said divalent cation is selected from the group comprising Mn²⁺ and Mg²⁺ ions.

32. The method of claim 20, wherein the method includes a plurality of said target nucleic acid sequences and a plurality of said second oligonucleotide molecules such that each of said second oligonucleotide molecules has a distinct 3′ region.

33. The method of claim 20, wherein the method includes at least ten said second oligonucleotide molecules or at least eleven said second oligonucleotide molecules, or at least twelve said second oligonucleotide molecules, or at least thirteen said second oligonucleotide molecules, or at least fourteen said second oligonucleotide molecules, or at least fifteen said second oligonucleotide molecules, or at least sixteen said second oligonucleotide molecules, or at least seventeen said second oligonucleotide molecules, or at least eighteen said second oligonucleotide molecules, or at least nineteen said second oligonucleotide molecules, or at least twenty said second oligonucleotide molecules, or at least twenty-one said second oligonucleotide molecules, or at least twenty-two said second oligonucleotide molecules, or at least twenty-three said second oligonucleotide molecules, or at least twenty-four said second oligonucleotide molecules, or at least twenty-five said second oligonucleotide molecules, or at least twenty-six said second oligonucleotide molecules, or at least twenty-seven said second, oligonucleotide molecules, or at least twenty-eight said second oligonucleotide molecules, or at least twenty-nine said second oligonucleotide molecules, or at least thirty said second oligonucleotide molecules, or at least thirty-one said second oligonucleotide molecules, or at least thirty-two said second oligonucleotide molecules, or at least thirty-three said second oligonucleotide molecules, or at least thirty-four said second oligonucleotide molecules, or at least thirty-five said second oligonucleotide molecules, or at least thirty-six said second oligonucleotide molecules, or at least thirty-seven said second oligonucleotide molecules, or at least thirty-eight said second oligonucleotide molecules, or at least thirty-nine said second oligonucleotide molecules, or at least forty said second oligonucleotide molecules, or at least forty-one said second oligonucleotide molecules, or at least forty-two said second oligonucleotide molecules, or at least forty-three said second oligonucleotide molecules, or at least forty-four said second oligonucleotide molecules, or at least forty-five said second oligonucleotide molecules, or at least forty-six said second oligonucleotide molecules, or at least forty-seven said second oligonucleotide molecules, or at least forty-eight said second oligonucleotide molecules, or at least forty-nine said second oligonucleotide molecules, or at least fifty said second oligonucleotide molecules, or at least sixty said second oligonucleotide molecules, or at least seventy said second oligonucleotide molecules, or at least eighty said second oligonucleotide molecules, or at least ninety said second oligonucleotide molecules, or at least one hundred said second oligonucleotide molecules, or at least one hundred and ten said second oligonucleotide molecules, or at least one hundred and twenty said second oligonucleotide molecules, or at least one hundred and thirty said second oligonucleotide molecules, or at least one hundred and forty said second oligonucleotide molecules, or at least one hundred and fifty said second oligonucleotide molecules, or at least one hundred and sixty said second oligonucleotide molecules, or at least one hundred and seventy said second oligonucleotide molecules, or at least one hundred and eighty said second oligonucleotide molecules, or at least one hundred and ninety said second oligonucleotide molecules, or at least two hundred said second oligonucleotide molecules.

34. The method of claim 20, wherein said 3′ portion of said second oligonucleotide molecules incorporates a fluorescent molecule, a radiolabelled nucleotide, digoxigenin, biotinylation and the like.

35. A composition comprising a cleavage structure, said cleavage structure comprising:

1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 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1 3 1 2 2 1 2 1 3 1 2 2 2 3 2 1 3 1 3 1 1 1 3 2 3 1 1 3 1 3 2 1 2 3 2 1 1 2 1 3 2 1 2 2 3 2 1 2 2 2 2 3 2 1 1 2 3 2 2 3 2 2 3 1 3 2 2 2 1 1 3 1 1 3 2 1 1 1 2 1 3 2 1 3 2 1 2 3 1 1 2 1 1 3 1 3 3 1 1 2 3 2 2 3 1 1 2 2 3 1 1 1 2 1 2 3 1 3 2 2 1 3 2 1 3 2 2 1 1 2 2 3 1 2 1 3 2 1 1 3 2 2 2 3 1 3 2 3 2 1 1 1 3 1 1 1 2 3 1 1 2 3 1 1 2 1 1 3 2 3 2 2 1 3 1 2 1 2 2 2 3 2 3 1 1 1 2 3 2 3 1 1 2 3 2 1 2 3 2 2 3 1 3 2 2 2 3 1 1 2 2 3 2 2 1 2 2 3 1 3 2 3 1 1 2 2 1 3 2 2 1 2 3 2 2 3 2 2 1 2 3 1 1 3 1 1 1 3 1 1 1 2 3 1 3 1 1 1 3 1 2 2 1 2 2 2 1 1 3 2 1 1 3 2 2 3 2 3 2 2 3 1 2 1 2 2 1 3 1 2 3 1 2 3 2 3 2 2 2 3 1 2 2 2 3 1 1 2 2 3 1 1 1 1 3 2 1 1 3 2 3 1 1 1 2 2 3 2 2 3 2 2 2 3 1 1 1 2 3 1 1 3 2 3 2 1 1 1 3 2 2 2 3 1 1 1 3 1 1 1 1 3 1 3 1 3 2 1 1 3 1 2 1 1 2 2 3 2 1 2 1 3 2 1 2 2 2 1 2 3 1 3 1 2 1 3 1 2 3 1 1 1 2 1 1 3 2 3 1 3 1 1 1 2 2 1 3 2 1 3 2 1 1 2 3 1 2 2 2 3 2 3 3 1 2 2 2 3 1 3 1 2 2 3 1 1 2 3 1 3 1 1 2 1 2 1 3 1 2 2 1 3 1 1 1 3 1 2 3 1 1 2 1 1 1 3 1 2 3 1 2 1 3 1 2 1 3 1 1 1 3 2 1 2 1 2 3 2 2 3 2 1 3 2 3 1 1 3 1 2 1 3 2 1 1 1 3 2 1 1 1 3 2 1 1 3 2 2 1 1 1 2 3 2 3 2 3 2 2 2 1 3 2 1 3 2 2 3 2 1 1 1 2 2 3 2 2 3 1 1 3 2 1 1 3 1 3 1 2 3 1 1 2 1 1 1 2 1 2 2 2 3 1 3 1 3 1 1 1 3 1 1 1 3 1 3 2 2 2 1 2 1 2 2 2 1 3 2 3 1 2 3 1 1 2 2 2 3 2 3 1 2 3 2 2 2 1 2 1 3 2 3 1 2 3 1 2 3 1 2 1 1 3 2 2 3 1 2 2 1 1 1 3 1 2 1 1 2 2 3 2 1 3 1 1 1 3 2 1 3 2 3 3 2 2 2 1 3 2 1 2 2 3 1 2 1 2 2 3 2 3 2 3 2 1 1 3 2 3 2 2 3 2 3 1 1 2 1 1 3 1 2 2 3 1 1 1 2 1 2 1 1 1 3 1 1 1 3 2 1 2 1 1 1 3 2 3 1 3 1 2 1 3 1 2 1 2 2 2 3 2 1 1 3 1 1 3 2 3 2 1 3 1 2 1 2 2 3 2 1 3 1 1 3 1 2 3 1 1 1 2 2 3 2 3 1 2 2 2 3 2 2 1 2 1 1 2 1 3 2 1 1 3 2 3 1 1 2 3 1 2 3 1 3 1 2 1 1 2 3 2 3 1 1 2 1 1 3 1 2 1 1 1 3 2 3 2 3 2 1 1 2 2 3 1 1 3 1 2 1 1 1 3 1 2 3 2 2 3 2 2 2 1 3 2 3 1 1 1 2 1 3 1 1 3 2 3 1 3 1 2 2 1 2 1 3 2 1 1 3 2 2 1 2 2 3 2 3 1 1 1 3 1 3 2 2 2 1 2 3 1 2 1 1 1 2 1 3 2 1 3 2 3 1 2 1 3 1 3 1 1 3 1 2 2 2 1 3 2 3 2 1 2 3 1 1 3 2 3 2 1 1 2 1 1 3 1 2 2 1 2 3 1 2 2 1 1 3 1 2 2 3 2 3 2 1 3 2 3 2 2 1 2 1 1 3 2 2 2 1 2 3 1 2 1 2 2 2 3 2 1 3 1 2 3 1 3 2 2 1 2 3 2 3 2 1 2 3 1 1 3 1 2 2 1 2 1 3 2 2 1 3 3 1 1 1 2 2 3 2 2 3 2 1 2 1 3 1 3 2 3 1 2 1 2 1 2 1 3 1 1 1 2 1 3 2 2 2 1 1 3 2 1 2 1 3 2 3 2 3 2 3 1 2 2 2 1 3 1 2 3 2 2 2 1 2 2 3 2 3 1 3 1 1 1 1 3 2 2 3 1 2 1 2 2 2 3 2 2 3 2 2 1 3 1 2 3 1 2 3 1 2 3 2 3 1 2 1 1 2 3 1 3 1 1 2 1 1 1 3 1 1 1 1 1 3 2 2 2 1 3 2 2 2 3 2 1 2 2 1 3 2 1 3 1 3 1 3 2 2 2 3 1 2 3 2 3 1 2 1 3 2 1 1 1 2 1 3 1 1 1 1 3 2 3 2 2 1 2 2 3 1 1 2 3 2 3 1 2 3 2 2 1 2 1 1 1 2 2 3 1 1 3 2 3 2 3 1 2 1 1 2 3 2 2 2 3 2 3 2 2 2 1 3 2 3 1 2 2 1 1 1 3 1 2 1 3 1 2 2 1 3 1 2 1 1 3 2 3 2 1 2 1 1 3 1 3 1 1 3 2 3 2 2 1 1 1 2 3 1 1 2 2 2 3 2 2 2 3 2 3 1 2 3 1 1 3 2 2 1 1 1 1 3 1 1 2 2 3 1 3 1 1 1 2 3 1 1 1 3 2 2 1 3 1 3 2 3 2 1 1 3 1 3 2 1 2 1 1 1 3 2 1 2 2 2 3 1 3 1 1 2 2 1 1 3 1 2 2 3 2 2 1 2 1 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 2 3 2 2 3 2 2 2 1 1 3 2 1 1 3 1 2 1 1 1 3 2 1 1 1 2 3 2 2 1 2 3 2 3 1 3 1 3 1 1 1 1 1 3 1 2 1 2 2 3 1 2 2 3 1 3 1 2 1 3 1 3 2 2 1 1 3 2 3 1 2 1 2 3 1 1 2 1 2 3 2 3 1 3 1 1 1 2 1 1 1 2 1 3 1 3 2 2 2 3 2 2 1 1 2 3 2 1 1 3 2 3 3 1 2 2 2 1 3 1 2 3 1 3 2 2 1 1 3 1 1 2 2 2 1 3 2 2 3 2 1 1 1 2 3 1 3 2 3 2 3 1 1 2 1 2 2 3 2 1 1 3 2 1 3 2 3 2 1 2 2 2 3 1 3 1 2 1 1 2 1 3 1 1 2 3 1 3 2 2 1 1 1 3 1 3 2 2 3 2 2 3 1 2 1 2 2 2 1 1 1 3 1 3 2 3 2 1 2 2 1 3 1 1 1 2 1 3 2 2 2 3 3 2 2 2 1 3 2 2 1 2 2 2 3 1 2 3 1 3 1 2 1 1 2 3 1 1 3 2 3 2 1 1 1 2 3 1 1 2 1 1 1 3 1 3 2 2 3 2 1 1 2 1 1 1 3 2 3 1 3 2 1 3 1 1 3 2 3 2 1 1 2 2 2 1 2 2 3 1 3 2 2 2 3 2 3 2 1 1 1 3 1 1 3 1 2 1 2 2 2 1 2 1 3 2 2 3 2 2 3 2 3 2 2 3 1 1 1 3 2 2 1 2 3 1 1 1 2 1 2 3 1 2 2 3 2 3 2 2 2 3 2 2 3 2 1 1 1 3 1 3 1 2 3 2 1 1 1 3 2 3 1 3 2 2 1 2 2 1 2 2 3 1 1 3 1 1 1 3 2 2 1 3 1 2 3 1 2 3 1 1 2 2 1 2 3 2 2 1 2 2 2 3 2 2 2 1 3 2 2 2 3 2 3 2 3 1 1 1 1 2 1 2 3 1 1 2 2 2 3 1 1 3 1 3 1 1 3 2 3 1 3 1 2 2 1 3 1 2 1 2 1 3 1 1 2 1 2 2 3 1 1 3 1 3 2 2 1 3 1 1 2 1 1 3 1 3 1 1 1 2 3 2 1 2 3 2 3 2 2 2 2 1 2 3 1 1 1 3 1 3 1 1 3 2 3 2 1 2 2 1 2 3 3 2 1 1 3 2 1 2 2 1 1 3 2 3 2 3 1 2 2 2 1 3 2 1 1 2 1 1 1 3 1 3 1 1 3 2 1 1 1 3 1 3 2 1 1 1 3 2 1 2 2 3 2 2 1 1 2 2 3 1 1 3 2 3 2 1 2 3 1 1 1 3 2 1 2 1 2 1 3 2 2 3 1 3 2 2 3 1 3 2 1 1 3 1 2 2 2 1 3 1 2 3 1 3 1 2 1 2 1 2 3 1 1 1 3 1 2 1 3 2 1 2 1 1 3 1 1 3 1 2 3 1 2 2 2 3 2 3 2 1 1 1 2 3 2 2 1 3 2 1 1 2 1 1 3 1 1 1 3 1 2 3 1 1 3 1 3 1 3 1 2 2 2 3 2 3 1 3 2 1 1 1 3 2 1 1 1 2 1 3 1 1 1 1 1 2 1 3 1 2 3 2 1 3 1 1 2 2 2 3 2 3 2 3 2 2 3 1 1 1 2 2 1 3 2 3 2 2 2 3 2 3 2 3 2 1 2 2 1 2 1 2 2 2 3 1 3 2 1 2 3 1 2 1 3 1 1 3 1 2 2 3 2 2 1 2 1 3 1 3 2 2 3 1 1 3 2 1 2 3 2 1 1 1 3 2 2 2 2 1 1 2 2 2 3 2 3 1 1 2 3 2 2 3 2 2 1 2 2 3 2 3 2 2 1 3 2 2 2 1 2 3 1 3 1 3 2 3 1 3 1 2 2 2 1 1 3 2 2 3 2 2 1 3 1 3 2 3 2 2 2 1 2 3 1 1 1 2 2 2 2 2 2 1 2 2 3 2 3 1 2 3 2 3 1 1 1 2 1 1 3 1 3 1 3 2 1 1 3 2 1 1 2 1 2 3 1 2 1 3 2 3 1 2 2 1 1 3 2 3 1 3 1 2 3 1 2 3 2 1 2 1 2 3 2 1 3 2 1 1 2 1 1 1 2 2 3 1 3 1 1 1 3 1 3 1 2 1 1 1 2 3 1 2 1 3 2 2 2 3 1 1 3 2 3 1 2 3 2 2 1 3 1 1 2 3 2 2 2 1 1 3 2 2 3 2 2 3 2 3 2 1 1 2 2 3 2 2 1 3 2 1 1 1 1 2 1 3 2 3 1 3 1 1 3 2 3 1 2 1 1 3 1 2 1 2 2 2 3 2 3 2 3 1 2 1 1 3 2 1 1 2 2 3 1 3 2 2 1 1 1 2 2 1 3 2 2 1 2 2 3 2 2 2 3 2 3 1 1 2 2 2 3 1 3 1 1 2 1 3 2 2 3 1 1 2 1 3 2 1 1 2 2 2 3 1 1 3 1 3 1 2 3 2 2 2 3 2 3 2 2 2 3 1 1 2 1 3 1 3 1 1 2 1 2 3 1 2 1 1 1 3 1 2 1 2 3 1 3 1 3 1 2 2 3 2 1 1 2 1 1 1 3 1 2 3 1 3 1 2 3 2 2 3 2 2 1 1 1 3 2 2 1 1 3 2 3 1 1 1 2 2 2 3 2 1 1 3 1 1 2 2 1 3 2 3 3 1 1 1 2 3 1 3 1 3 2 2 1 2 2 3 1 2 1 3 2 2 2 1 2 2 2 3 2 1 1 1 2 3 1 3 1 2 1 2 1 3 2 3 2 2 1 3 3 2 2 1 1 2 2 3 2 3 1 2 1 2 2 2 3 1 2 2 1 3 2 3 1 3 1 3 2 3 2 2 3 1 2 1 1 1 3 1 2 3 2 2 2 1 2 1 1 1 2 2 3 2 3 1 3 1 1 1 2 2 3 1 2 1 1 3 1 1 3 1 2 2 1 1 1 3 1 3 1 1 2 2 3 1 3 1 1 3 1 3 1 1 1 2 2 2 3 2 2 1 3 1 1 3 1 1 2 2 3 1 1 2 3 2 1 2 3 2 1 3 2 2 1 1 3 1 2 1 2 3 2 3 2 3 1 2 3 2 2 2 1 1 2 3 1 3 2 2 1 2 3 2 2 3 2 1 1 2 1 3 1 1 1 2 2 3 2 2 1 3 1 2 1 1 3 2 2 2 1 3 1 3 1 2 2 3 1 3 1 1 1 2 3 1 3 2 1 1 2 1 1 3 1 3 2 1 2 2 2 3 1 1 3 2 2 3 2 2 2 1 1 3 2 3 2 1 1 2 3 1 2 2 2 3 2 2 1 3 2 2 3 1 1 3 1 1 3 1 2 2 3 2 2 1 2 2 3 2 2 3 1 1 2 1 2 1 3 1 1 1 3 1 2 2 1 1 1 3 1 3 2 3 1 1 2 3 2 1 1 1 2 2 3 2 2 1 3 1 1 1 2 2 2 3 1 3 2 3 2 3 1 2 2 3 2 2 1 3 2 3 2 3 2 2 1 2 2 3 1 2 2 1 2 3 3 1 3 1 1 2 2 1 2 3 2 3 2 3 1 1 2 1 2 1 3 1 1 1 2 2 3 1 2 2 3 1 2 1 1 1 3 2 1 1 1 3 1 3 2 3 2 1 3 2 3 2 3 2 1 1 1 2 2 3 1 1 2 1 2 3 2 2 1 1 2 3 1 1 1 3 2 1 1 1 3 1 1 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 2 1 3 2 2 3 1 1 3 1 1 2 1 3 1 1 1 3 1 1 1 3 3 2 3 2 1 1 2 1 1 3 1 3 2 3 1 1 2 1 3 2 1 1 2 2 2 1 2 2 3 1 1 1 2 1 1 3 1 3 1 3 1 2 2 2 3 2 3 1 1 2 3 1 3 1 1 1 3 1 1 3 1 1 3 2 2 1 1 3 1 2 2 2 1 1 3 1 3 2 3 1 3 2 1 2 1 2 2 3 2 2 1 1 1 3 1 1 2 2 2 3 2 1 1 1 3 2 3 1 2 3 1 2 3 2 1 1 3 1 2 1 3 1 2 3 2 2 1 2 3 2 3 1 2 3 1 1 1 2 1 2 3 2 1 2 3 2 1 3 1 1 2 1 1 1 3 2 3 2 2 1 1 1 3 2 3 2 2 1 1 1 1 3 1 3 2 1 2 3 2 3 2 3 2 1 2 3 1 2 1 2 2 2 1 1 1 3 1 2 1 1 3 1 3 2 2 1 3 2 1 1 1 2 2 3 2 3 1 1 3 1 1 2 2 1 3 1 3 1 1 2 1 1 3 2 3 2 3 1 2 1 3 1 2 1 1 3 1 1 1 3 2 3 1 1 1 2 3 2 1 1 1 2 2 3 3 1 2 3 1 1 1 3 1 2 3 2 2 2 1 1 1 3 2 2 2 3 2 2 1 3 2 3 2 1 1 3 2 1 1 2 1 1 3 2 2 2 3 1 3 1 1 1 3 2 2 3 1 3 1 1 2 2 1 3 1 1 2 2 2 3 1 2 1 1 1 3 2 2 1 1 3 1 1 1 2 2 2 3 2 1 2 3 2 3 2 2 3 2 2 3 1 3 1 1 3 1 2 2 2 1 3 1 2 3 1 1 1 2 3 1 3 2 2 2 2 1 1 3 2 2 2 3 1 3 1 2 1 1 1 3 1 2 3 1 2 1 2 3 2 3 1 3 1 2 1 3 2 2 2 3 2 1 1 2 1 2 3 2 2 2 3 2 1 3 2 2 2 3 1 1 1 2 2 3 2 1 1 3 2 2 2 3 1 2 3 1 2 1 3 1 1 2 2 3 1 2 2 1 1 2 3 1 2 3 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 2 1 1 2 1 1 3 2 1 2 2 3 1 1 3 1 2 1 1 2 3 1 2 3 2 1 1 2 3 2 1 1 3 2 1 3 2 3 2 2 3 1 1 1 2 2 2 3 1 2 3 1 3 1 3 1 2 1 2 3 2 2 1 2 3 2 3 2 1 1 1 3 2 1 2 1 3 2 2 2 1 2 3 2 2 1 3 2 3 1 1 2 1 1 3 2 3 1 1 1 2 1 3 1 1 2 3 1 1 2 3 1 1 1 3 1 1 1 3 1 2 2 3 2 1 1 2 1 1 3 2 1 3 1 3 1 1 2 3 1 1 1 2 1 3 2 3 2 2 1 1 1 2 3 1 3 2 3 2 3 2 1 3 1 2 1 1 1 3 1 2 3 2 3 1 1 2 2 1 2 3 1 2 3 1 2 1 3 2 1 2 1 2 3 2 3 2 3 2 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 2 1 3 1 1 1 2 3 2 2 2 3 1 1 3 1 2 1 1 1 2 2 2 3 2 1 3 1 3 1 3 1 1 1 2 2 2 3 2 2 3 2 1 3 1 1 2 1 1 3 1 2 2 1 3 2 1 1 3 2 3 2 1 3 1 2 3 1 2 2 2 1 3 1 3 1 1 1 2 1 2 3 1 3 2 1 3 1 1 1 1 2 1 3 1 3 2 1 2 3 2 2 3 2 2 2 1 2 3 1 3 1 1 3 1 2 3 1 2 3 1 1 3 1 3 2 2 2 1 2 2 3 2 1 1 1 2 1 1 3 2 1 1 1 3 1 1 3 1 1 3 1 1 1 2 3 2 3 2 2 1 2 2 3 1 1 3 1 1 2 2 1 1 3 2 3 2 2 2 1 3 2 3 2 1 1 3 1 1 1 3 1 1 2 3 2 2 3 1 2 2 2 1 2 3 1 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 2 3 1 2 2 3 1 1 1 2 1 1 2 3 1 2 1 1 2 2 3 2 2 3 1 3 1 3 1 3 2 1 1 2 3 2 2 2 3 2 2 3 1 1 1 3 2 3 2 1 1 1 3 2 1 2 1 2 2 3 1 3 2 2 1 2 1 2 3 1 3 1 1 1 3 2 3 2 1 1 2 2 2 2 3 2 3 1 3 1 1 1 3 1 1 3 2 1 2 1 2 1 3 1 1 2 3 2 1 1 3 2 2 2 1 3 1 3 2 2 1 2 1 3 1 3 2 2 2 1 3 1 2 1 3 1 2 1 3 1 2 1 1 3 2 2 1 1 2 2 3 1 1 3 1 3 1 3 1 2 3 1 2 2 3 2 2 2 1 2 3 2 1 2 2 1 2 3 1 1 1 3 2 2 1 1 3 1 1 1 2 2 3 2 1 3 2 3 1 2 1 3 2 2 2 3 1 2 1 2 2 3 2 2 2 3 2 3 1 3 2 3 2 1 2 1 1 2 2 2 3 2 1 3 1 1 1 3 2 2 3 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 2 1 1 3 2 2 2 1 2 1 3 1 2 3 1 1 1 1 3 1 2 1 1 1 3 2 3 1 3 2 2 3 1 2 2 2 1 3 1 2 3 1 2 1 2 2 3 2 1 1 3 1 2 1 2 3 2 2 3 2 1 1 1 3 3 2 1 1 3 1 3 2 3 2 1 2 2 3 2 1 1 3 2 2 1 1 2 2 3 2 3 2 3 1 2 2 1 3 2 1 1 2 3 1 1 3 2 1 2 2 2 1 3 2 1 1 3 1 1 1 3 1 2 2 1 1 3 2 3 2 2 1 3 2 1 1 1 3 2 1 3 1 1 1 3 2 2 3 1 1 1 2 2 3 1 2 2 1 2 3 2 1 1 3 1 3 1 1 3 2 2 3 1 3 2 1 1 2 3 2 1 2 2 2 3 2 2 1 1 3 1 1 1 2 1 3 2 1 3 1 2 1 1 3 2 3 1 1 2 1 1 3 2 1 1 1 2 2 3 1 1 1 3 2 3 2 1 2 1 3 2 3 1 1 3 1 2 3 2 1 2 3 2 2 2 1 2 2 3 2 2 3 2 3 2 1 1 2 2 2 1 3 1 1 2 1 2 1 3 2 3 1 1 3 1 3 1 2 1 3 3 2 2 1 2 3 1 1 1 3 1 3 2 1 2 3 2 3 2 2 1 1 1 2 2 1 2 2 1 2 3 2 3 1 1 3 1 1 3 1 1 2 3 1 2 2 1 3 2 1 1 2 1 1 3 2 2 3 1 1 3 1 3 1 1 2 2 3 2 2 3 2 2 3 1 2 3 2 2 2 3 1 2 3 2 1 1 2 2 3 2 2 1 1 1 3 3 2 3 1 1 1 3 1 2 2 2 3 1 3 2 2 2 3 2 1 2 1 1 2 1 3 1 3 1 1 2 1 2 1 3 1 2 2 3 1 3 1 2 2 2 3 2 2 2 2 2 3 1 3 1 2 3 2 3 1 2 3 1 2 1 1 1 3 2 2 1 1 3 2 2 3 2 1 1 1 2 2 3 2 1 3 2 1 1 1 3 1 1 3 2 1 3 2 3 2 2 1 2 3 1 2 3 2 2 3 2 2 2 3 2 1 2 2 1 2 1 2 2 1 2 2 3 2 3 2 1 3 1 2 3 2 1 2 2 1 1 3 1 3 3 2 2 1 3 1 1 1 3 1 2 2 2 1 3 1 1 3 2 2 1 3 2 2 2 2 3 2 3 2 1 2 2 1 1 3 1 3 1 3 2 3 1 1 1 2 1 2 3 2 2 2 1 1 3 1 2 1 3 1 1 1 3 1 3 2 3 1 2 2 2 1 1 1 2 3 1 3 1 1 1 2 1 3 1 2 1 3 2 2 1 2 2 3 2 3 2 3 1 1 2 2 3 1 1 2 1 1 3 1 1 2 2 2 3 2 2 3 2 3 1 1 2 1 1 3 1 2 2 3 1 1 2 2 1 3 2 3 1 3 2 1 1 3 1 1 2 3 2 2 2 3 1 3 1 3 1 2 2 2 1 3 2 1 1 1 3 1 1 3 2 2 2 1 3 1 1 2 1 3 1 1 1 2 3 2 3 2 2 2 3 1 2 1 2 1 1 3 2 1 1 3 2 3 2 2 1 1 3 1 2 2 2 3 1 3 3 2 1 3 2 3 1 1 2 1 1 3 2 2 1 3 2 3 2 2 1 1 2 1 1 1 3 2 3 2 3 2 2 1 1 1 3 2 1 1 1 2 3 2 1 3 1 2 1 3 1 3 1 2 3 2 2 2 1 2 3 2 2 3 2 3 1 1 2 2 1 1 1 3 2 2 3 1 1 2 1 2 2 3 1 2 3 1 2 1 1 3 1 1 3 1 2 3 1 1 2 3 2 3 1 3 1 2 3 2 2 2 1 3 1 1 2 1 1 2 1 1 2 1 1 2 3 1 2 3 2 1 1 3 2 2 2 3 1 3 2 2 2 3 1 1 1 3 1 3 2 3 1 1 2 1 3 1 1 1 2 1 1 3 1 3 1 1 1 1 2 1 1 1 3 2 2 1 2 2 3 1 3 1 3 1 3 2 2 2 1 3 1 3 2 1 3 2 3 2 2 3 2 1 3 2 2 2 1 3 2 1 2 1 2 1 3 2 1 1 3 1 1 2 3 2 1 2 2 1 3 1 2 1 2 2 2 3 2 3 3 1 2 1 1 1 2 3 2 2 2 3 1 2 1 1 1 3 2 1 3 2 2 3 1 2 1 3 2 1 2 3 2 1 2 3 2 3 2 3 1 1 3 1 2 2 2 1 1 2 3 1 1 2 3 2 1 3 1 3 2 3 1 2 2 1 3 2 2 2 1 1 3 2 1 3 2 1 2 2 2 1 3 2 3 1 2 3 2 1 1 3 1 1 2 1 1 3 1 1 2 2 3 2 1 2 2 3 1 1 3 1 1 3 1 1 2 1 2 3 2 2 2 1 2 1 3 1 1 2 2 3 1 3 1 3 1 1 3 2 2 1 1 3 1 1 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 1 3 1 1 2 2 1 2 2 2 1 1 1 3 1 1 1 3 2 1 2 2 3 2 1 1 3 1 3 2 3 1 1 1 2 2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 1 3 2 2 2 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1 2 2 2 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1 1 3 1 3 1 1 2 1 1 2 3 2 1 3 1 3 1 2 1 2 1 1 3 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 1 1 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1 2 2 3 2 3 2 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 3 1 2 2 3 1 2 3 2 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 1 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 2 2 1 1 1 2 3 1 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 1 2 3 1 1 2 1 1 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 2 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 2 2 2 3 1 1 2 3 1 1 1 2 2 3 1 2 3 1 2 1 3 1 2 3 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 3 1 1 1 3 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 2 2 2 1 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 2 1 1 1 3 2 3 1 1 1 3 1 2 2 2 3 1 1 1 2 3 1 2 3 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 2 2 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 2 2 3 2 3 1 1 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 2 2 3 2 3 2 2 3 1 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 2 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 2 2 3 2 1 2 2 2 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 1 3 1 1 1 2 1 2 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 2 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 3 2 2 3 2 1 1 2 2 2 3 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 2 2 3 2 3 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1 3 2 3 1 3 1 2 2 2 1 3 1 1 3 1 2 3 2 2 1 2 2 1 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 3 2 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2

wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that:

for any pair of sequences of the set:

M1≦16, M2≦13, M3 S 20., M4 s 16, and M5≦19, where:

M1 is the maximum number of matches for any alignment in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment;

M3 is the maximum number of matches for any alignment having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein

the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:

for each of (i) to (iv):

(i) m=6, mm=6, og=0 and eg=6,

(ii) m=6, mm=6, og=5 and eg=1,

(iii) m=6, mm=2, og=5 and eg=1, and

(iv) m=6, mm=6, og=6 and eg=0,

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and

wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv)

c) detecting said non-target cleavage products.

36. The composition of claim 35, wherein one or more of said first and second oligonucleotides contain a 3′-terminal dideoxynucleotide.

37. The composition of claim 35, wherein the composition includes a plurality of said target nucleic acid molecules and a plurality of said second oligonucleotide molecules such that each of said second oligonucleotide molecules has a distinct 3′ region.

38. The composition of claim 35, wherein the composition includes at least ten, or twenty, or thirty, or forty, or fifty, or sixty, or seventy, or eighty, or ninety, or one hundred, or one hundred and ten, or one hundred and twenty, or one hundred and thirty, or one hundred and forty, or one hundred and fity, or one hundred and sixty said second oligonucelotide molecules, or comprising one hundred and seventy said second oligonucleotide molecules, or comprising one hundred and eighty said second oligonucleotide molecules, or comprising one hundred and ninety said second oligonucleotide molecules, or comprising two hundred said second oligonucleotide molecules, or comprising two hundred and twenty said second oligonucleotide molecules, or comprising two hundred and forty said second oligonucleotide molecules, or comprising two hundred and sixty said second oligonucleotide molecules, or comprising two hundred and eighty said second oligonucleotide molecules, or comprising three hundred said second oligonucleotide molecules, or comprising four hundred said second oligonucleotide molecules, or comprising five hundred said second oligonucleotide molecules, or comprising six hundred said second oligonucleotide molecules, or comprising seven hundred said second oligonucleotide molecules, or comprising eight hundred said second oligonucleotide molecules, or comprising nine hundred said second oligonucleotide molecules, or comprising one thousand said second oligonucleotide molecules, or comprising eleven hundred said second oligonucleotide molecules.

39. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products, the method comprising:

a) providing:

i) a cleavage means,

iv) a second oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central portion of said second oligonucleotide having a sequence complimentary to said second region of said target nucleic acid, and said 3′ portion of said second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides based on a following group of sequences,

1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 1 2 3 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 3 2 3 1 3 1 2 1 2 1 3 1 2 2 2 1 3 1 1 1 3 2 1 1 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 2 1 3 2 1 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 1 2 2 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 1 3 1 2 1 1 3 1 1 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 3 2 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 2 2 2 3 2 3 1 1 3 1 2 3 1 1 3 2 1 2 2 2 3 2 1 2 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 2 1 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 3 2 2 1 2 2 2 3 2 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 3 1 1 2 1 3 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 1 2 2 3 2 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 2 3 1 3 1 1 2 3 2 1 1 1 3 1 1 2 3 2 2 2 1 2 2 3 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 3 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 3 1 2 2 3 1 2 1 2 2 2 3 1 1 2 3 2 2 2 3 2 2 2 3 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 2 1 2 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 1 1 1 2 2 2 3 1 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 2 2 3 2 2 3 2 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 1 2 3 2 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 2 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 1 1 3 1 1 1 2 3 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 1 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 1 2 1 3 1 2 3 2 1 1 3 1 3 1 1 1 2 2 3 2 3 1 1 1 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 3 1 3 2 1 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 1 1 2 3 2 1 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 1 2 3 2 2 3 1 3 2 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 3 1 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 2 1 2 2 3 2 1 3 1 1 3 2 1 1 1 3 2 2 1 3 1 1 3 2 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 2 3 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 1 2 1 1 3 1 1 1 2 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 3 1 2 1 2 1 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 3 1 2 2 1 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 2 1 2 3 2 3 2 3 2 2 3 2 2 2 1 3 2 3 2 2 1 2 2 1 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 3 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 2 1 3 2 1 2 2 1 3 2 1 1 3 2 1 2 3 2 3 1 2 2 3 2 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 1 1 2 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 3 1 2 2 3 1 1 2 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 2 1 1 3 2 2 1 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 2 2 2 1 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 3 1 2 1 3 1 2 2 2 1 3 1 1 2 3 1 1 2 2 1 1 3 2 3 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 1 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 2 3 2 2 2 1 2 3 2 1 3 2 3 2 1 3 1 2 2 3 1 1 2 2 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 2 1 3 2 1 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 2 1 3 2 2 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 3 2 1 1 2 3 1 2 1 1 2 3 1 1 3 2 3 2 1 2 1 2 1 3 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 2 1 1 2 3 1 1 3 1 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 2 1 3 2 2 1 3 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 3 1 1 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 1 3 2 2 1 2 1 1 3 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 2 3 2 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 3 1 3 2 2 1 1 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 1 3 2 1 2 1 2 1 3 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 2 1 2 3 2 1 1 1 3 1 1 1 3 2 3 1 1 1 3 1 1 3 1 1 2 3 1 1 2 3 2 1 3 1 1 1 2 3 1 1 2 3 2 2 3 1 1 1 1 1 2 2 3 1 1 2 1 3 2 3 2 3 2 3 1 3 2 2 2 1 1 2 1 3 1 2 1 2 2 3 2 2 2 3 1 2 2 1 1 2 3 1 1 3 1 3 1 1 1 3 2 2 3 2 1 1 1 3 2 2 3 1 1 3 1 2 1 1 1 3 3 2 2 1 1 3 1 3 1 2 2 1 2 3 1 3 1 2 3 2 1 2 2 1 1 3 1 1 3 1 2 1 2 1 1 3 1 1 3 1 2 2 3 1 1 2 2 3 3 2 1 3 1 1 1 2 2 2 3 1 1 2 2 3 1 2 3 2 3 1 1 1 1 1 3 1 3 2 1 3 1 2 2 3 1 2 1 1 3 2 1 2 1 2 3 1 2 3 1 2 1 2 1 3 2 1 3 2 3 1 1 3 1 1 1 2 1 1 3 2 1 3 1 2 1 1 2 3 1 2 3 1 3 1 1 1 2 3 1 1 3 1 2 1 1 2 3 2 3 1 1 1 3 2 1 2 2 2 3 2 3 1 2 1 2 1 3 2 1 1 2 1 1 3 1 3 1 1 2 2 3 1 2 1 2 3 1 1 3 1 2 3 2 1 1 3 2 3 2 1 2 2 2 1 3 2 1 3 1 1 2 3 1 1 3 2 2 1 2 3 2 2 1 3 1 2 2 2 3 2 2 3 1 3 1 2 2 3 1 2 1 3 2 2 2 3 2 1 2 3 1 1 3 1 3 1 2 1 3 2 1 2 2 2 3 1 3 1 1 1 2 3 2 2 1 2 3 2 1 2 2 2 1 3 2 1 3 2 2 1 2 3 2 3 1 3 1 1 2 3 2 3 2 2 2 3 1 2 2 2 1 1 3 2 1 2 3 2 2 2 3 2 2 2 1 2 1 3 1 1 2 3 2 1 2 3 3 1 3 2 1 2 1 2 1 3 1 1 3 1 1 1 3 1 1 1 2 2 2 3 1 2 3 1 3 2 3 1 1 3 2 1 1 1 2 3 2 1 3 2 2 1 2 2 2 2 1 1 3 1 1 3 2 3 1 3 2 2 1 2 2 3 2 3 1 2 1 2 1 2 3 1 1 1 2 3 1 3 1 1 2 1 2 2 3 2 2 3 2 2 2 3 3 1 2 2 1 1 2 3 1 2 2 1 2 3 2 3 1 1 2 2 3 1 2 3 3 1 1 1 2 3 2 2 1 1 1 3 1 2 1 2 3 1 1 1 3 2 1 3 2 1 2 2 3 2 2 3 1 2 2 2 3 1 2 1 2 2 1 3 2 3 2 3 2 2 2 1 2 3 2 2 2 3 2 3 2 1 2 3 2 1 1 3 2 1 3 2 1 1 2 2 3 1 1 1 3 1 1 2 2 3 2 3 2 3 1 1 2 2 3 1 2 3 1 3 2 2 2 3 1 1 2 2 2 3 2 2 2 3 1 3 2 1 1 2 3 1 2 3 2 1 2 1 1 2 3 1 2 3 2 3 2 3 2 1 1 1 2 2 1 2 3 2 3 1 3 1 3 1 1 3 1 1 2 2 2 3 2 2 2 1 2 2 3 2 3 1 2 1 1 1 3 2 1 2 2 3 2 2 3 1 2 1 3 1 1 1 3 1 1 3 2 1 3 1 1 2 1 3 1 1 1 3 2 2 1 1 2 1 3 1 2 2 3 2 3 2 1 3 2 2 1 1 3 1 3 2 2 3 2 2 2 1 1 2 2 1 3 2 1 3 2 1 1 3 2 2 3 2 2 1 3 1 1 2 1 3 2 2 1 1 2 2 2 3 1 1 3 2 1 2 1 1 2 3 1 1 2 3 2 3 2 3 2 1 3 1 1 1 2 2 3 2 1 3 2 1 2 2 2 3 1 3 1 3 1 1 2 3 2 1 2 1 2 3 2 2 1 1 2 3 1 3 1 2 3 2 2 3 2 1 2 1 2 2 2 3 1 2 1 1 3 1 3 1 1 2 3 1 1 3 1 1 3 2 2 2 3 1 1 2 1 3 2 3 2 1 1 2 3 1 1 2 1 2 3 1 2 3 3 2 1 3 2 2 2 3 2 3 1 1 2 1 3 1 1 2 2 1 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 2 1 1 2 2 2 3 2 3 2 3 1 1 3 1 1 3 1 2 2 3 2 2 3 1 3 2 2 1 1 2 1 3 1 2 1 1 1 3 1 2 2 1 2 3 2 1 3 2 3 1 2 3 2 1 1 1 2 3 2 2 3 1 1 2 2 2 1 3 1 2 3 2 1 3 1 2 1 2 3 1 1 2 3 2 3 1 2 1 3 1 1 3 2 3 2 1 2 2 1 1 3 2 1 1 3 2 2 1 2 1 2 3 1 1 2 2 1 2 3 1 3 1 1 3 1 1 2 1 3 1 3 2 2 2 2 3 2 2 1 2 3 1 1 3 2 3 1 2 2 2 3 2 2 2 3 2 3 2 1 1 1 3 1 2 2 3 2 3 2 2 1 2 1 2 3 1 1 1 2 3 2 2 3 2 3 1 2 1 3 2 1 3 2 2 1 3 1 2 1 2 2 2 3 2 3 1 1 2 2 1 1 3 1 2 1 1 1 3 1 1 3 1 3 1 1 3 2 1 3 1 2 2 3 2 1 3 1 1 2 3 1 1 2 2 2 3 2 1 3 2 1 2 1 1 1 2 1 1 3 1 3 1 3 1 3 1 1 2 3 1 2 2 2 1 3 2 1 1 2 2 1 2 3 2 3 1 1 2 1 3 1 2 2 3 2 2 3 1 1 3 2 2 1 1 3 1 2 2 2 1 2 3 2 3 1 2 1 3 2 1 3 1 3 2 2 2 1 1 1 3 1 2 1 3 2 3 2 2 2 3 2 2 3 2 3 2 2 1 2 1 2 2 3 1 2 2 2 1 2 3 1 1 3 1 3 2 1 2 1 3 2 3 1 1 1 2 2 2 3 1 2 3 1 3 2 1 3 2 2 2 1 1 3 1 3 1 1 2 1 1 1 3 2 2 3 2 2 2 3 1 2 3 2 2 2 3 1 1 2 3 3 1 2 2 3 2 3 1 2 3 1 1 2 1 1 2 3 2 2 1 2 2 3 1 3 1 2 3 1 1 3 1 1 1 2 1 2 3 1 2 1 2 3 1 1 2 1 3 2 2 1 1 1 3 2 2 1 2 2 3 1 1 3 2 3 1 1 3 2 2 3 1 2 2 3 2 1 1 3 1 1 1 2 1 3 1 3 1 2 2 2 3 2 3 2 2 3 1 3 1 2 2 3 1 3 2 2 2 1 1 3 2 1 2 2 1 3 1 2 2 1 3 2 3 1 2 1 1 2 1 3 1 1 2 3 1 2 1 1 1 2 3 2 3 3 1 2 1 1 2 1 3 2 3 1 1 2 2 2 3 1 3 2 2 3 2 1 2 1 3 1 2 1 2 2 2 3 2 1 3 2 1 3 1 1 1 3 2 1 2 3 2 3 2 2 1 2 3 1 1 2 3 2 2 3 1 1 2 2 2 3 1 1 2 3 2 1 2 3 1 1 1 3 1 2 2 2 1 3 2 2 3 2 3 1 3 1 2 1 2 1 1 1 2 1 3 1 3 1 1 3 2 2 1 2 3 1 2 3 2 3 1 2 1 2 2 1 3 2 3 1 3 1 1 1 2 3 2 2 2 1 1 2 3 2 3 1 2 2 3 1 1 3 1 1 2 1 2 3 2 3 1 1 1 2 2 1 3 2 2 2 3 3 2 2 2 3 1 2 1 3 2 2 2 1 1 2 3 1 3 2 1 2 2 3 1 3 2 2 3 2 1 1 3 2 1 1 2 3 1 2 1 1 1 3 2 1 2 3 1 2 1 1 3 1 3 2 1 3 2 1 1 2 2 3 2 2 3 2 2 2 1 3 1 2 2 2 3 1 3 1 3 1 3 2 1 2 3 2 1 2 3 1 2 2 1 2 2 1 2 2 3 1 2 2 3 2 3 1 1 2 2 1 3 1 2 1 3 1 1 3 1 3 1 2 2 1 3 2 1 2 2 2 1 3 2 1 3 2 1 1 2 1 3 1 3 2 1 2 3 2 1 2 2 1 3 1 3 1 2 1 2 2 3 1 1 1 3 2 3 2 1 2 3 2 3 1 1 1 3 2 1 1 2 3 1 2 1 1 1 2 3 1 3 3 2 1 1 2 2 1 3 2 1 1 2 3 1 2 2 2 3 1 1 2 3 1 3 3 2 2 2 1 2 2 3 2 1 1 1 3 1 2 3 2 1 1 3 2 3 1 1 2 1 3 2 1 3 1 1 2 2 3 2 2 3 2 2 1 1 1 3 1 1 2 3 2 1 2 2 3 2 2 1 3 2 2 1 2 3 2 1 3 2 3 2 3 2 1 1 3 1 3 2 3 1 1 1 3 2 2 1 2 1 2 3 1 1 1 3 2 1 2 1 1 2 1 2 1 3 1 1 3 2 2 3 1 2 3 1 3 2 2 2 1 2 3 1 2 2 2 1 3 1 1 3 2 1 1 3 1 1 2 1 1 3 2 3 1 3 2 1 2 3 2 3 2 1 2 1 1 3 1 2 1 2 2 2 3 2 1 1 3 1 1 3 2 1 3 1 1 3 1 3 2 2 3 2 1 2 2 3 2 2 1 2 1 1 3 2 3 2 3 2 2 1 2 2 1 3 2 2 2 1 1 3 2 2 1 3 1 3 2 1 1 1 2 1 2 1 3 2 3 1 2 3 2 3 1 1 1 2 2 3 1 1 2 3 2 2 1 3 1 3 1 1 2 1 3 1 3 2 3 1 2 2 1 2 1 3 2 2 3 1 1 3 2 3 1 3 2 2 1 1 2 3 1 2 2 2 3 2 1 1 1 2 1 1 2 3 2 1 1 1 3 2 1 1 1 3 1 1 1 3 2 3 1 2 3 1 3 2 2 1 3 2 2 1 2 3 1 2 3 1 1 2 1 2 2 3 2 3 2 1 1 1 1 2 3 1 3 2 2 1 3 1 3 2 1 3 1 1 2 2 1 2 3 2 3 1 2 1 2 1 3 1 1 3 1 2 2 1 3 2 2 1 3 2 3 1 2 1 1 2 1 3 2 2 2 3 2 2 3 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 1 1 2 1 3 2 2 1 3 2 1 3 2 1 1 3 1 3 2 1 2 3 1 1 2 2 2 3 2 1 2 2 3 2 3 1 1 3 2 2 2 1 3 2 1 3 2 1 3 2 1 1 3 1 1 3 1 3 1 1 2 2 1 3 1 2 2 1 1 1 1 2 3 2 3 2 2 1 2 3 2 1 2 3 2 1 1 1 2 1 3 2 3 3 1 1 2 2 1 3 2 2 1 3 1 3 2 1 1 1 2 2 3 2 2 2 3 3 1 1 1 2 2 3 1 1 3 1 2 1 3 2 1 1 3 1 1 1 2 3 1 3 2 3 2 1 2 2 1 2 3 2 3 1 2 2 2 1 2 3 1 2 1 3 1 2 1 2 2 1 2 3 1 3 1 1 1 3 2 2 3 1 1 2 1 3 2 1 3 2 1 2 3 2 1 2 2 3 2 1 2 2 3 1 3 2 1 3 1 2 3 1 1 3 2 3 1 2 2 3 1 1 2 1 3 2 1 3 1 2 2 3 2 2 2 1 1 1 3 2 1 1 3 2 2 3 2 2 2 3 1 2 2 3 1 1 1 2 2 2 3 3 1 1 3 2 2 2 3 1 2 2 2 1 1 3 2 2 2 1 1 3 1 1 3 3 1 3 1 1 3 1 2 1 1 1 2 3 1 2 1 2 2 3 2 2 1 2 3 1 2 3 1 2 3 1 3 2 2 3 2 2 1 1 2 1 3 2 2 1 3 2 2 2 1 2 3 1 2 1 2 2 2 3 1 1 3 1 3 2 3 2 2 1 1 3 1 3 1 3 1 2 3 1 2 2 1 1 1 3 2 3 1 2 2 2 1 2 3 1 1 1 2 1 3 2 2 1 1 3 1 3 2 3 1 2 3 1 3 1 1 2 1 1 1 2 2 2 1 2 2 3 2 2 1 3 1 2 1 1 1 3 1 3 2 2 3 1 3 1 3 1 1 2 1 2 2 3 1 2 1 3 2 2 3 1 1 3 2 2 3 1 1 2 1 3 2 3 2 1 1 1 3 2 3 2 1 3 1 2 2 3 2 1 1 1 2 1 3 2 1 3 2 3 1 2 1 2 3 1 2 2 2 3 1 1 2 1 2 2 3 2 3 2 1 2 2 3 1 1 2 2 1 3 1 1 2 1 3 2 3 1 3 1 1 2 3 1 2 1 2 3 1 3 1 2 1 3 1 1 3 2 2 2 1 1 2 3 2 3 1 1 3 1 1 3 2 1 1 3 2 1 2 1 1 1 3 2 1 1 1 2 3 2 2 2 1 1 3 2 3 2 3 1 2 1 1 3 1 1 1 3 1 2 1 3 1 2 1 2 2 3 2 2 3 1 1 2 3 2 3 2 2 2 1 1 1 3 1 3 1 3 1 1 2 1 1 2 3 1 2 3 1 3 1 2 3 1 2 2 1 2 2 3 1 2 1 3 1 3 1 1 1 3 1 3 1 3 1 1 2 2 3 2 1 2 2 1 1 1 2 3 2 1 2 1 1 2 3 1 3 1 2 1 2 3 2 2 2 3 2 3 1 1 1 2 1 3 1 2 1 1 3 1 2 2 3 1 2 2 3 2 3 2 2 2 3 2 2 2 3 1 2 3 1 2 1 1 2 1 3 1 1 3 1 3 1 1 2 3 1 1 3 1 2 3 1 1 2 1 1 3 2 2 3 2 3 1 1 2 3 2 2 2 1 1 3 1 2 3 1 1 1 3 1 1 1 3 2 3 2 1 3 1 1 2 1 2 2 2 3 2 2 1 1 1 2 3 2 1 2 3 2 1 3 2 1 1 2 2 3 1 3 2 1 3 2 1 3 2 3 2 3 1 1 3 2 2 1 2 2 2 3 2 2 1 2 1 3 2 3 1 1 2 3 2 2 2 3 2 1 1 1 3 1 3 2 2 2 1 1 3 1 2 1 1 1 2 3 1 3 1 1 2 2 3 1 3 2 1 1 2 2 3 2 2 3 1 2 3 1 3 1 1 1 2 2 3 2 2 2 1 1 3 2 3 2 2 2 1 1 1 2 1 1 3 2 1 3 2 3 2 3 1 3 2 1 1 2 1 3 2 1 2 1 2 3 1 1 1 2 1 2 3 2 3 1 2 1 3 2 1 1 3 1 3 1 1 2 2 3 2 1 1 3 1 3 2 3 1 2 2 1 2 1 3 1 2 3 1 2 1 3 1 3 2 1 1 3 1 1 2 3 1 1 1 3 1 3 1 2 1 1 2 1 2 1 1 3 2 1 1 3 2 1 3 1 2 3 2 2 1 1 1 3 1 3 1 2 1 1 1 2 1 3 1 1 1 3 1 1 2 2 3 2 1 3 1 3 2 1 3 2 1 2 1 3 1 2 2 2 1 1 3 2 3 1 1 3 1 3 1 3 2 2 1 2 3 1 1 2 3 2 2 2 3 2 1 1 1 2 3 2 1 2 1 3 1 2 1 3 1 1 1 2 1 3 1 1 2 3 1 3 2 1 3 2 3 1 1 1 2 1 2 3 2 2 3 1 1 2 2 1 2 3 2 1 3 1 3 1 1 1 3 2 1 1 1 3 2 1 3 2 1 1 1 2 2 3 1 3 1 3 2 1 3 2 2 3 1 1 2 2 2 3 2 1 1 1 3 2 3 2 2 2 1 2 1 3 2 3 2 3 2 1 1 2 1 2 1 2 3 1 2 2 2 3 1 3 1 2 3 1 3 1 1 2 3 2 1 1 1 1 2 1 2 2 3 1 2 1 2 3 2 3 2 2 3 2 3 1 1 3 2 1 1 3 2 3 1 3 1 2 2 1 2 3 1 3 2 1 2 2 3 1 2 2 2 1 2 2 3 2 1 2 2 2 1 3 1 2 1 3 2 3 1 3 1 2 2 1 2 3 1 2 1 3 1 1 1 2 3 1 1 1 3 1 2 1 3 1 2 1 3 1 1 3 3 1 2 2 3 2 1 2 1 2 3 2 1 1 1 3 2 1 3 2 2 2 1 3 2 1 2 3 1 1 2 3 2 2 1 2 2 3 2 3 2 3 2 2 3 1 2 2 3 1 2 1 2 2 1 3 2 1 3 1 3 2 1 1 3 2 1 2 1 2 2 3 2 3 1 3 1 2 3 1 1 2 2 2 3 2 3 2 2 1 2 3 1 2 1 2 2 1 2 3 1 1 2 3 1 1 3 2 1 1 1 3 1 3 1 2 3 2 1 1 3 1 3 2 3 1 1 2 2 2 3 2 2 3 2 1 1 2 2 2 3 2 2 2 1 3 1 1 1 2 2 3 2 1 3 1 3 2 2 1 1 2 2 3 2 3 2 1 3 2 3 2 2 1 1 2 3 1 1 1 3 2 2 3 2 3 1 1 2 1 1 2 2 3 2 3 1 2 2 2 3 2 2 1 1 3 1 1 3 1 2 2 1 1 2 3 1 3 2 1 3 2 1 2 2 3 2 1 1 1 3 2 1 2 1 1 1 3 1 3 2 3 1 2 2 3 2 2 3 2 1 2 1 3 2 2 1 2 2 3 2 3 2 1 3 1 2 2 3 2 1 3 2 2 2 1 1 2 3 2 2 1 1 3 1 1 2 3 1 2 3 1 1 1 2 1 1 3 1 1 1 2 2 3 1 3 2 1 3 1 3 1 2 1 2 3 1 2 3 1 2 1 2 2 2 3 2 2 3 2 1 2 3 2 3 2 2 2 2 1 3 1 3 2 2 2 3 1 2 2 1 3 2 1 2 3 2 2 2 3 1 1 2 1 1 3 1 3 1 2 2 3 2 3 1 2 3 1 3 1 1 1 2 1 1 1 2 3 1 1 2 1 3 1 1 2 1 3 1 3 1 1 2 3 2 1 3 1 3 2 1 3 2 1 3 2 1 1 2 2 2 3 1 1 2 3 2 2 2 3 1 1 1 3 2 3 1 3 2 1 1 2 2 3 1 2 2 3 1 2 2 3 2 2 1 1 3 1 1 2 1 1 2 3 2 2 2 1 3 2 3 2 3 2 2 2 3 1 1 1 1 2 1 2 3 1 1 1 3 2 1 3 1 3 1 1 1 3 2 3 2 2 1 2 2 3 1 3 2 2 1 2 2 3 2 1 2 2 2 1 3 2 2 2 3 1 1 3 2 1 3 2 2 3 1 3 2 2 2 1 1 1 3 2 2 3 1 1 1 3 1 1 2 1 1 1 3 1 3 2 3 1 2 3 2 1 1 1 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1 3 1 2 2 1 2 1 3 1 2 2 2 3 2 1 3 1 3 1 1 1 3 2 3 1 1 3 1 3 2 1 2 3 2 1 1 2 1 3 2 1 2 2 3 2 1 2 2 2 2 3 2 1 1 2 3 2 2 3 2 2 3 1 3 2 2 2 1 1 3 1 1 3 2 1 1 1 2 1 3 2 1 3 2 1 2 3 1 1 2 1 1 3 1 3 3 1 1 2 3 2 2 3 1 1 2 2 3 1 1 1 2 1 2 3 1 3 2 2 1 3 2 1 3 2 2 1 1 2 2 3 1 2 1 3 2 1 1 3 2 2 2 3 1 3 2 3 2 1 1 1 3 1 1 1 2 3 1 1 2 3 1 1 2 1 1 3 2 3 2 2 1 3 1 2 1 2 2 2 3 2 3 1 1 1 2 3 2 3 1 1 2 3 2 1 2 3 2 2 3 1 3 2 2 2 3 1 1 2 2 3 2 2 1 2 2 3 1 3 2 3 1 1 2 2 1 3 2 2 1 2 3 2 2 3 2 2 1 2 3 1 1 3 1 1 1 3 1 1 1 2 3 1 3 1 1 1 3 1 2 2 1 2 2 2 1 1 3 2 1 1 3 2 2 3 2 3 2 2 3 1 2 1 2 2 1 3 1 2 3 1 2 3 2 3 2 2 2 3 1 2 2 2 3 1 1 2 2 3 1 1 1 1 3 2 1 1 3 2 3 1 1 1 2 2 3 2 2 3 2 2 2 3 1 1 1 2 3 1 1 3 2 3 2 1 1 1 3 2 2 2 3 1 1 1 3 1 1 1 1 3 1 3 1 3 2 1 1 3 1 2 1 1 2 2 3 2 1 2 1 3 2 1 2 2 2 1 2 3 1 3 1 2 1 3 1 2 3 1 1 1 2 1 1 3 2 3 1 3 1 1 1 2 2 1 3 2 1 3 2 1 1 2 3 1 2 2 2 3 2 3 3 1 2 2 2 3 1 3 1 2 2 3 1 1 2 3 1 3 1 1 2 1 2 1 3 1 2 2 1 3 1 1 1 3 1 2 3 1 1 2 1 1 1 3 1 2 3 1 2 1 3 1 2 1 3 1 1 1 3 2 1 2 1 2 3 2 2 3 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1 3 1 2 3 1 3 2 2 1 2 3 2 3 2 1 2 3 1 1 3 1 2 2 1 2 1 3 2 2 1 3 3 1 1 1 2 2 3 2 2 3 2 1 2 1 3 1 3 2 3 1 2 1 2 1 2 1 3 1 1 1 2 1 3 2 2 2 1 1 3 2 1 2 1 3 2 3 2 3 2 3 1 2 2 2 1 3 1 2 3 2 2 2 1 2 2 3 2 3 1 3 1 1 1 1 3 2 2 3 1 2 1 2 2 2 3 2 2 3 2 2 1 3 1 2 3 1 2 3 1 2 3 2 3 1 2 1 1 2 3 1 3 1 1 2 1 1 1 3 1 1 1 1 1 3 2 2 2 1 3 2 2 2 3 2 1 2 2 1 3 2 1 3 1 3 1 3 2 2 2 3 1 2 3 2 3 1 2 1 3 2 1 1 1 2 1 3 1 1 1 1 3 2 3 2 2 1 2 2 3 1 1 2 3 2 3 1 2 3 2 2 1 2 1 1 1 2 2 3 1 1 3 2 3 2 3 1 2 1 1 2 3 2 2 2 3 2 3 2 2 2 1 3 2 3 1 2 2 1 1 1 3 1 2 1 3 1 2 2 1 3 1 2 1 1 3 2 3 2 1 2 1 1 3 1 3 1 1 3 2 3 2 2 1 1 1 2 3 1 1 2 2 2 3 2 2 2 3 2 3 1 2 3 1 1 3 2 2 1 1 1 1 3 1 1 2 2 3 1 3 1 1 1 2 3 1 1 1 3 2 2 1 3 1 3 2 3 2 1 1 3 1 3 2 1 2 1 1 1 3 2 1 2 2 2 3 1 3 1 1 2 2 1 1 3 1 2 2 3 2 2 1 2 1 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 2 3 2 2 3 2 2 2 1 1 3 2 1 1 3 1 2 1 1 1 3 2 1 1 1 2 3 2 2 1 2 3 2 3 1 3 1 3 1 1 1 1 1 3 1 2 1 2 2 3 1 2 2 3 1 3 1 2 1 3 1 3 2 2 1 1 3 2 3 1 2 1 2 3 1 1 2 1 2 3 2 3 1 3 1 1 1 2 1 1 1 2 1 3 1 3 2 2 2 3 2 2 1 1 2 3 2 1 1 3 2 3 3 1 2 2 2 1 3 1 2 3 1 3 2 2 1 1 3 1 1 2 2 2 1 3 2 2 3 2 1 1 1 2 3 1 3 2 3 2 3 1 1 2 1 2 2 3 2 1 1 3 2 1 3 2 3 2 1 2 2 2 3 1 3 1 2 1 1 2 1 3 1 1 2 3 1 3 2 2 1 1 1 3 1 3 2 2 3 2 2 3 1 2 1 2 2 2 1 1 1 3 1 3 2 3 2 1 2 2 1 3 1 1 1 2 1 3 2 2 2 3 3 2 2 2 1 3 2 2 1 2 2 2 3 1 2 3 1 3 1 2 1 1 2 3 1 1 3 2 3 2 1 1 1 2 3 1 1 2 1 1 1 3 1 3 2 2 3 2 1 1 2 1 1 1 3 2 3 1 3 2 1 3 1 1 3 2 3 2 1 1 2 2 2 1 2 2 3 1 3 2 2 2 3 2 3 2 1 1 1 3 1 1 3 1 2 1 2 2 2 1 2 1 3 2 2 3 2 2 3 2 3 2 2 3 1 1 1 3 2 2 1 2 3 1 1 1 2 1 2 3 1 2 2 3 2 3 2 2 2 3 2 2 3 2 1 1 1 3 1 3 1 2 3 2 1 1 1 3 2 3 1 3 2 2 1 2 2 1 2 2 3 1 1 3 1 1 1 3 2 2 1 3 1 2 3 1 2 3 1 1 2 2 1 2 3 2 2 1 2 2 2 3 2 2 2 1 3 2 2 2 3 2 3 2 3 1 1 1 1 2 1 2 3 1 1 2 2 2 3 1 1 3 1 3 1 1 3 2 3 1 3 1 2 2 1 3 1 2 1 2 1 3 1 1 2 1 2 2 3 1 1 3 1 3 2 2 1 3 1 1 2 1 1 3 1 3 1 1 1 2 3 2 1 2 3 2 3 2 2 2 2 1 2 3 1 1 1 3 1 3 1 1 3 2 3 2 1 2 2 1 2 3 3 2 1 1 3 2 1 2 2 1 1 3 2 3 2 3 1 2 2 2 1 3 2 1 1 2 1 1 1 3 1 3 1 1 3 2 1 1 1 3 1 3 2 1 1 1 3 2 1 2 2 3 2 2 1 1 2 2 3 1 1 3 2 3 2 1 2 3 1 1 1 3 2 1 2 1 2 1 3 2 2 3 1 3 2 2 3 1 3 2 1 1 3 1 2 2 2 1 3 1 2 3 1 3 1 2 1 2 1 2 3 1 1 1 3 1 2 1 3 2 1 2 1 1 3 1 1 3 1 2 3 1 2 2 2 3 2 3 2 1 1 1 2 3 2 2 1 3 2 1 1 2 1 1 3 1 1 1 3 1 2 3 1 1 3 1 3 1 3 1 2 2 2 3 2 3 1 3 2 1 1 1 3 2 1 1 1 2 1 3 1 1 1 1 1 2 1 3 1 2 3 2 1 3 1 1 2 2 2 3 2 3 2 3 2 2 3 1 1 1 2 2 1 3 2 3 2 2 2 3 2 3 2 3 2 1 2 2 1 2 1 2 2 2 3 1 3 2 1 2 3 1 2 1 3 1 1 3 1 2 2 3 2 2 1 2 1 3 1 3 2 2 3 1 1 3 2 1 2 3 2 1 1 1 3 2 2 2 2 1 1 2 2 2 3 2 3 1 1 2 3 2 2 3 2 2 1 2 2 3 2 3 2 2 1 3 2 2 2 1 2 3 1 3 1 3 2 3 1 3 1 2 2 2 1 1 3 2 2 3 2 2 1 3 1 3 2 3 2 2 2 1 2 3 1 1 1 2 2 2 2 2 2 1 2 2 3 2 3 1 2 3 2 3 1 1 1 2 1 1 3 1 3 1 3 2 1 1 3 2 1 1 2 1 2 3 1 2 1 3 2 3 1 2 2 1 1 3 2 3 1 3 1 2 3 1 2 3 2 1 2 1 2 3 2 1 3 2 1 1 2 1 1 1 2 2 3 1 3 1 1 1 3 1 3 1 2 1 1 1 2 3 1 2 1 3 2 2 2 3 1 1 3 2 3 1 2 3 2 2 1 3 1 1 2 3 2 2 2 1 1 3 2 2 3 2 2 3 2 3 2 1 1 2 2 3 2 2 1 3 2 1 1 1 1 2 1 3 2 3 1 3 1 1 3 2 3 1 2 1 1 3 1 2 1 2 2 2 3 2 3 2 3 1 2 1 1 3 2 1 1 2 2 3 1 3 2 2 1 1 1 2 2 1 3 2 2 1 2 2 3 2 2 2 3 2 3 1 1 2 2 2 3 1 3 1 1 2 1 3 2 2 3 1 1 2 1 3 2 1 1 2 2 2 3 1 1 3 1 3 1 2 3 2 2 2 3 2 3 2 2 2 3 1 1 2 1 3 1 3 1 1 2 1 2 3 1 2 1 1 1 3 1 2 1 2 3 1 3 1 3 1 2 2 3 2 1 1 2 1 1 1 3 1 2 3 1 3 1 2 3 2 2 3 2 2 1 1 1 3 2 2 1 1 3 2 3 1 1 1 2 2 2 3 2 1 1 3 1 1 2 2 1 3 2 3 3 1 1 1 2 3 1 3 1 3 2 2 1 2 2 3 1 2 1 3 2 2 2 1 2 2 2 3 2 1 1 1 2 3 1 3 1 2 1 2 1 3 2 3 2 2 1 3 3 2 2 1 1 2 2 3 2 3 1 2 1 2 2 2 3 1 2 2 1 3 2 3 1 3 1 3 2 3 2 2 3 1 2 1 1 1 3 1 2 3 2 2 2 1 2 1 1 1 2 2 3 2 3 1 3 1 1 1 2 2 3 1 2 1 1 3 1 1 3 1 2 2 1 1 1 3 1 3 1 1 2 2 3 1 3 1 1 3 1 3 1 1 1 2 2 2 3 2 2 1 3 1 1 3 1 1 2 2 3 1 1 2 3 2 1 2 3 2 1 3 2 2 1 1 3 1 2 1 2 3 2 3 2 3 1 2 3 2 2 2 1 1 2 3 1 3 2 2 1 2 3 2 2 3 2 1 1 2 1 3 1 1 1 2 2 3 2 2 1 3 1 2 1 1 3 2 2 2 1 3 1 3 1 2 2 3 1 3 1 1 1 2 3 1 3 2 1 1 2 1 1 3 1 3 2 1 2 2 2 3 1 1 3 2 2 3 2 2 2 1 1 3 2 3 2 1 1 2 3 1 2 2 2 3 2 2 1 3 2 2 3 1 1 3 1 1 3 1 2 2 3 2 2 1 2 2 3 2 2 3 1 1 2 1 2 1 3 1 1 1 3 1 2 2 1 1 1 3 1 3 2 3 1 1 2 3 2 1 1 1 2 2 3 2 2 1 3 1 1 1 2 2 2 3 1 3 2 3 2 3 1 2 2 3 2 2 1 3 2 3 2 3 2 2 1 2 2 3 1 2 2 1 2 3 3 1 3 1 1 2 2 1 2 3 2 3 2 3 1 1 2 1 2 1 3 1 1 1 2 2 3 1 2 2 3 1 2 1 1 1 3 2 1 1 1 3 1 3 2 3 2 1 3 2 3 2 3 2 1 1 1 2 2 3 1 1 2 1 2 3 2 2 1 1 2 3 1 1 1 3 2 1 1 1 3 1 1 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 2 1 3 2 2 3 1 1 3 1 1 2 1 3 1 1 1 3 1 1 1 3 3 2 3 2 1 1 2 1 1 3 1 3 2 3 1 1 2 1 3 2 1 1 2 2 2 1 2 2 3 1 1 1 2 1 1 3 1 3 1 3 1 2 2 2 3 2 3 1 1 2 3 1 3 1 1 1 3 1 1 3 1 1 3 2 2 1 1 3 1 2 2 2 1 1 3 1 3 2 3 1 3 2 1 2 1 2 2 3 2 2 1 1 1 3 1 1 2 2 2 3 2 1 1 1 3 2 3 1 2 3 1 2 3 2 1 1 3 1 2 1 3 1 2 3 2 2 1 2 3 2 3 1 2 3 1 1 1 2 1 2 3 2 1 2 3 2 1 3 1 1 2 1 1 1 3 2 3 2 2 1 1 1 3 2 3 2 2 1 1 1 1 3 1 3 2 1 2 3 2 3 2 3 2 1 2 3 1 2 1 2 2 2 1 1 1 3 1 2 1 1 3 1 3 2 2 1 3 2 1 1 1 2 2 3 2 3 1 1 3 1 1 2 2 1 3 1 3 1 1 2 1 1 3 2 3 2 3 1 2 1 3 1 2 1 1 3 1 1 1 3 2 3 1 1 1 2 3 2 1 1 1 2 2 3 3 1 2 3 1 1 1 3 1 2 3 2 2 2 1 1 1 3 2 2 2 3 2 2 1 3 2 3 2 1 1 3 2 1 1 2 1 1 3 2 2 2 3 1 3 1 1 1 3 2 2 3 1 3 1 1 2 2 1 3 1 1 2 2 2 3 1 2 1 1 1 3 2 2 1 1 3 1 1 1 2 2 2 3 2 1 2 3 2 3 2 2 3 2 2 3 1 3 1 1 3 1 2 2 2 1 3 1 2 3 1 1 1 2 3 1 3 2 2 2 2 1 1 3 2 2 2 3 1 3 1 2 1 1 1 3 1 2 3 1 2 1 2 3 2 3 1 3 1 2 1 3 2 2 2 3 2 1 1 2 1 2 3 2 2 2 3 2 1 3 2 2 2 3 1 1 1 2 2 3 2 1 1 3 2 2 2 3 1 2 3 1 2 1 3 1 1 2 2 3 1 2 2 1 1 2 3 1 2 3 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 2 1 1 2 1 1 3 2 1 2 2 3 1 1 3 1 2 1 1 2 3 1 2 3 2 1 1 2 3 2 1 1 3 2 1 3 2 3 2 2 3 1 1 1 2 2 2 3 1 2 3 1 3 1 3 1 2 1 2 3 2 2 1 2 3 2 3 2 1 1 1 3 2 1 2 1 3 2 2 2 1 2 3 2 2 1 3 2 3 1 1 2 1 1 3 2 3 1 1 1 2 1 3 1 1 2 3 1 1 2 3 1 1 1 3 1 1 1 3 1 2 2 3 2 1 1 2 1 1 3 2 1 3 1 3 1 1 2 3 1 1 1 2 1 3 2 3 2 2 1 1 1 2 3 1 3 2 3 2 3 2 1 3 1 2 1 1 1 3 1 2 3 2 3 1 1 2 2 1 2 3 1 2 3 1 2 1 3 2 1 2 1 2 3 2 3 2 3 2 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 2 1 3 1 1 1 2 3 2 2 2 3 1 1 3 1 2 1 1 1 2 2 2 3 2 1 3 1 3 1 3 1 1 1 2 2 2 3 2 2 3 2 1 3 1 1 2 1 1 3 1 2 2 1 3 2 1 1 3 2 3 2 1 3 1 2 3 1 2 2 2 1 3 1 3 1 1 1 2 1 2 3 1 3 2 1 3 1 1 1 1 2 1 3 1 3 2 1 2 3 2 2 3 2 2 2 1 2 3 1 3 1 1 3 1 2 3 1 2 3 1 1 3 1 3 2 2 2 1 2 2 3 2 1 1 1 2 1 1 3 2 1 1 1 3 1 1 3 1 1 3 1 1 1 2 3 2 3 2 2 1 2 2 3 1 1 3 1 1 2 2 1 1 3 2 3 2 2 2 1 3 2 3 2 1 1 3 1 1 1 3 1 1 2 3 2 2 3 1 2 2 2 1 2 3 1 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 2 3 1 2 2 3 1 1 1 2 1 1 2 3 1 2 1 1 2 2 3 2 2 3 1 3 1 3 1 3 2 1 1 2 3 2 2 2 3 2 2 3 1 1 1 3 2 3 2 1 1 1 3 2 1 2 1 2 2 3 1 3 2 2 1 2 1 2 3 1 3 1 1 1 3 2 3 2 1 1 2 2 2 2 3 2 3 1 3 1 1 1 3 1 1 3 2 1 2 1 2 1 3 1 1 2 3 2 1 1 3 2 2 2 1 3 1 3 2 2 1 2 1 3 1 3 2 2 2 1 3 1 2 1 3 1 2 1 3 1 2 1 1 3 2 2 1 1 2 2 3 1 1 3 1 3 1 3 1 2 3 1 2 2 3 2 2 2 1 2 3 2 1 2 2 1 2 3 1 1 1 3 2 2 1 1 3 1 1 1 2 2 3 2 1 3 2 3 1 2 1 3 2 2 2 3 1 2 1 2 2 3 2 2 2 3 2 3 1 3 2 3 2 1 2 1 1 2 2 2 3 2 1 3 1 1 1 3 2 2 3 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 2 1 1 3 2 2 2 1 2 1 3 1 2 3 1 1 1 1 3 1 2 1 1 1 3 2 3 1 3 2 2 3 1 2 2 2 1 3 1 2 3 1 2 1 2 2 3 2 1 1 3 1 2 1 2 3 2 2 3 2 1 1 1 3 3 2 1 1 3 1 3 2 3 2 1 2 2 3 2 1 1 3 2 2 1 1 2 2 3 2 3 2 3 1 2 2 1 3 2 1 1 2 3 1 1 3 2 1 2 2 2 1 3 2 1 1 3 1 1 1 3 1 2 2 1 1 3 2 3 2 2 1 3 2 1 1 1 3 2 1 3 1 1 1 3 2 2 3 1 1 1 2 2 3 1 2 2 1 2 3 2 1 1 3 1 3 1 1 3 2 2 3 1 3 2 1 1 2 3 2 1 2 2 2 3 2 2 1 1 3 1 1 1 2 1 3 2 1 3 1 2 1 1 3 2 3 1 1 2 1 1 3 2 1 1 1 2 2 3 1 1 1 3 2 3 2 1 2 1 3 2 3 1 1 3 1 2 3 2 1 2 3 2 2 2 1 2 2 3 2 2 3 2 3 2 1 1 2 2 2 1 3 1 1 2 1 2 1 3 2 3 1 1 3 1 3 1 2 1 3 3 2 2 1 2 3 1 1 1 3 1 3 2 1 2 3 2 3 2 2 1 1 1 2 2 1 2 2 1 2 3 2 3 1 1 3 1 1 3 1 1 2 3 1 2 2 1 3 2 1 1 2 1 1 3 2 2 3 1 1 3 1 3 1 1 2 2 3 2 2 3 2 2 3 1 2 3 2 2 2 3 1 2 3 2 1 1 2 2 3 2 2 1 1 1 3 3 2 3 1 1 1 3 1 2 2 2 3 1 3 2 2 2 3 2 1 2 1 1 2 1 3 1 3 1 1 2 1 2 1 3 1 2 2 3 1 3 1 2 2 2 3 2 2 2 2 2 3 1 3 1 2 3 2 3 1 2 3 1 2 1 1 1 3 2 2 1 1 3 2 2 3 2 1 1 1 2 2 3 2 1 3 2 1 1 1 3 1 1 3 2 1 3 2 3 2 2 1 2 3 1 2 3 2 2 3 2 2 2 3 2 1 2 2 1 2 1 2 2 1 2 2 3 2 3 2 1 3 1 2 3 2 1 2 2 1 1 3 1 3 3 2 2 1 3 1 1 1 3 1 2 2 2 1 3 1 1 3 2 2 1 3 2 2 2 2 3 2 3 2 1 2 2 1 1 3 1 3 1 3 2 3 1 1 1 2 1 2 3 2 2 2 1 1 3 1 2 1 3 1 1 1 3 1 3 2 3 1 2 2 2 1 1 1 2 3 1 3 1 1 1 2 1 3 1 2 1 3 2 2 1 2 2 3 2 3 2 3 1 1 2 2 3 1 1 2 1 1 3 1 1 2 2 2 3 2 2 3 2 3 1 1 2 1 1 3 1 2 2 3 1 1 2 2 1 3 2 3 1 3 2 1 1 3 1 1 2 3 2 2 2 3 1 3 1 3 1 2 2 2 1 3 2 1 1 1 3 1 1 3 2 2 2 1 3 1 1 2 1 3 1 1 1 2 3 2 3 2 2 2 3 1 2 1 2 1 1 3 2 1 1 3 2 3 2 2 1 1 3 1 2 2 2 3 1 3 3 2 1 3 2 3 1 1 2 1 1 3 2 2 1 3 2 3 2 2 1 1 2 1 1 1 3 2 3 2 3 2 2 1 1 1 3 2 1 1 1 2 3 2 1 3 1 2 1 3 1 3 1 2 3 2 2 2 1 2 3 2 2 3 2 3 1 1 2 2 1 1 1 3 2 2 3 1 1 2 1 2 2 3 1 2 3 1 2 1 1 3 1 1 3 1 2 3 1 1 2 3 2 3 1 3 1 2 3 2 2 2 1 3 1 1 2 1 1 2 1 1 2 1 1 2 3 1 2 3 2 1 1 3 2 2 2 3 1 3 2 2 2 3 1 1 1 3 1 3 2 3 1 1 2 1 3 1 1 1 2 1 1 3 1 3 1 1 1 1 2 1 1 1 3 2 2 1 2 2 3 1 3 1 3 1 3 2 2 2 1 3 1 3 2 1 3 2 3 2 2 3 2 1 3 2 2 2 1 3 2 1 2 1 2 1 3 2 1 1 3 1 1 2 3 2 1 2 2 1 3 1 2 1 2 2 2 3 2 3 3 1 2 1 1 1 2 3 2 2 2 3 1 2 1 1 1 3 2 1 3 2 2 3 1 2 1 3 2 1 2 3 2 1 2 3 2 3 2 3 1 1 3 1 2 2 2 1 1 2 3 1 1 2 3 2 1 3 1 3 2 3 1 2 2 1 3 2 2 2 1 1 3 2 1 3 2 1 2 2 2 1 3 2 3 1 2 3 2 1 1 3 1 1 2 1 1 3 1 1 2 2 3 2 1 2 2 3 1 1 3 1 1 3 1 1 2 1 2 3 2 2 2 1 2 1 3 1 1 2 2 3 1 3 1 3 1 1 3 2 2 1 1 3 1 1 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 1 3 1 1 2 2 1 2 2 2 1 1 1 3 1 1 1 3 2 1 2 2 3 2 1 1 3 1 3 2 3 1 1 1 2 2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 1 3 2 2 2 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1 2 2 2 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1 1 3 1 3 1 1 2 1 1 2 3 2 1 3 1 3 1 2 1 2 1 1 3 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 1 1 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1 2 2 3 2 3 2 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 3 1 2 2 3 1 2 3 2 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 1 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 2 2 1 1 1 2 3 1 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 1 2 3 1 1 2 1 1 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 2 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 2 2 2 3 1 1 2 3 1 1 1 2 2 3 1 2 3 1 2 1 3 1 2 3 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 3 1 1 1 3 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 2 2 2 1 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 2 1 1 1 3 2 3 1 1 1 3 1 2 2 2 3 1 1 1 2 3 1 2 3 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 2 2 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 2 2 3 2 3 1 1 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 2 2 3 2 3 2 2 3 1 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 2 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 2 2 3 2 1 2 2 2 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 1 3 1 1 1 2 1 2 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 2 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 3 2 2 3 2 1 1 2 2 2 3 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 2 2 3 2 3 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1 3 2 3 1 3 1 2 2 2 1 3 1 1 3 1 2 3 2 2 1 2 2 1 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 3 2 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2

for any pair of sequences of the set:

M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19, where:

M1 is the maximum number of matches for any alignment in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment;

M3 is the maximum number of matches for any alignment having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein:

the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:

for each of (i) to (iv):

(i) m=6, mm=6, og=0 and eg=6,

(ii) m=6, mm=6, og=5 and eg=1,

(iii) m=6, mm=2, og=5 and eg=1, and

(iv) m=6, mm=6, og=6 and eg=0,

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and

wherein the maximum score is determined separately for each of (i), (ii)., (iii) and (iv).

c) detecting said non-target cleavage products.

40. The composition of claim 39, wherein one or more of said first and second oligonucleotides contain a 3′-terminal dideoxynucleotide.

41. The composition of claim 39, wherein the composition includes a plurality of said target nucleic acid sequences and a plurality of said second oligonucleotide molecules such that each of said second oligonucleotide molecules has a distinct 3′ region.

43. The composition of claim 39, wherein the composition includes at least ten, or twenty, or thirty, or forty, or fifty, or sixty, or seventy, or eighty, or ninety, or one hundred, or one hundred and ten, or one hundred and twenty, or one hundred and thirty, or one hundred and forty, or one hundred and fity, or one hundred and sixty said second oligonucelotide molecules, or comprising one hundred and seventy said second oligonucleotide molecules, or comprising one hundred and eighty said second oligonucleotide molecules, or comprising one hundred and ninety said second oligonucleotide molecules, or comprising two hundred said second oligonucleotide molecules, or comprising two hundred and twenty said second oligonucleotide molecules, or comprising two hundred and forty said second oligonucleotide molecules, or comprising two hundred and sixty said second oligonucleotide molecules, or comprising two hundred and eighty said second oligonucleotide molecules, or comprising three hundred said second oligonucleotide molecules, or comprising four hundred said second oligonucleotide molecules, or comprising five hundred said second oligonucleotide molecules, or comprising six hundred said second oligonucleotide molecules, or comprising seven hundred said second oligonucleotide molecules, or comprising eight hundred said second oligonucleotide molecules, or comprising nine hundred said second oligonucleotide molecules, or comprising one thousand said second oligonucleotide molecules, or comprising eleven hundred said second oligonucleotide molecules.

44. A method of analyzing a biological sample comprising a plurality of target nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule, the method comprising:

a) providing:

i) a cleavage means,

1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 1 2 3 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 3 2 3 1 3 1 2 1 2 1 3 1 2 2 2 1 3 1 1 1 3 2 1 1 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 2 1 3 2 1 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 1 2 2 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 1 3 1 2 1 1 3 1 1 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 3 2 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 2 2 2 3 2 3 1 1 3 1 2 3 1 1 3 2 1 2 2 2 3 2 1 2 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 2 1 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 3 2 2 1 2 2 2 3 2 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 3 1 1 2 1 3 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 1 2 2 3 2 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 2 3 1 3 1 1 2 3 2 1 1 1 3 1 1 2 3 2 2 2 1 2 2 3 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 3 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 3 1 2 2 3 1 2 1 2 2 2 3 1 1 2 3 2 2 2 3 2 2 2 3 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 2 1 2 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 1 1 1 2 2 2 3 1 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 2 2 3 2 2 3 2 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 1 2 3 2 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 2 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 1 1 3 1 1 1 2 3 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 1 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 1 2 1 3 1 2 3 2 1 1 3 1 3 1 1 1 2 2 3 2 3 1 1 1 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 3 1 3 2 1 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 1 1 2 3 2 1 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 1 2 3 2 2 3 1 3 2 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 3 1 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 2 1 2 2 3 2 1 3 1 1 3 2 1 1 1 3 2 2 1 3 1 1 3 2 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 2 3 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 1 2 1 1 3 1 1 1 2 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 3 1 2 1 2 1 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 3 1 2 2 1 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 2 1 2 3 2 3 2 3 2 2 3 2 2 2 1 3 2 3 2 2 1 2 2 1 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 3 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 2 1 3 2 1 2 2 1 3 2 1 1 3 2 1 2 3 2 3 1 2 2 3 2 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 1 1 2 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 3 1 2 2 3 1 1 2 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 2 1 1 3 2 2 1 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 2 2 2 1 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 3 1 2 1 3 1 2 2 2 1 3 1 1 2 3 1 1 2 2 1 1 3 2 3 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 1 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 2 3 2 2 2 1 2 3 2 1 3 2 3 2 1 3 1 2 2 3 1 1 2 2 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 2 1 3 2 1 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 2 1 3 2 2 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 3 2 1 1 2 3 1 2 1 1 2 3 1 1 3 2 3 2 1 2 1 2 1 3 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 2 1 1 2 3 1 1 3 1 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 2 1 3 2 2 1 3 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 3 1 1 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 1 3 2 2 1 2 1 1 3 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 2 3 2 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 3 1 3 2 2 1 1 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 1 3 2 1 2 1 2 1 3 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 2 1 2 3 2 1 1 1 3 1 1 1 3 2 3 1 1 1 3 1 1 3 1 1 2 3 1 1 2 3 2 1 3 1 1 1 2 3 1 1 2 3 2 2 3 1 1 1 1 1 2 2 3 1 1 2 1 3 2 3 2 3 2 3 1 3 2 2 2 1 1 2 1 3 1 2 1 2 2 3 2 2 2 3 1 2 2 1 1 2 3 1 1 3 1 3 1 1 1 3 2 2 3 2 1 1 1 3 2 2 3 1 1 3 1 2 1 1 1 3 3 2 2 1 1 3 1 3 1 2 2 1 2 3 1 3 1 2 3 2 1 2 2 1 1 3 1 1 3 1 2 1 2 1 1 3 1 1 3 1 2 2 3 1 1 2 2 3 3 2 1 3 1 1 1 2 2 2 3 1 1 2 2 3 1 2 3 2 3 1 1 1 1 1 3 1 3 2 1 3 1 2 2 3 1 2 1 1 3 2 1 2 1 2 3 1 2 3 1 2 1 2 1 3 2 1 3 2 3 1 1 3 1 1 1 2 1 1 3 2 1 3 1 2 1 1 2 3 1 2 3 1 3 1 1 1 2 3 1 1 3 1 2 1 1 2 3 2 3 1 1 1 3 2 1 2 2 2 3 2 3 1 2 1 2 1 3 2 1 1 2 1 1 3 1 3 1 1 2 2 3 1 2 1 2 3 1 1 3 1 2 3 2 1 1 3 2 3 2 1 2 2 2 1 3 2 1 3 1 1 2 3 1 1 3 2 2 1 2 3 2 2 1 3 1 2 2 2 3 2 2 3 1 3 1 2 2 3 1 2 1 3 2 2 2 3 2 1 2 3 1 1 3 1 3 1 2 1 3 2 1 2 2 2 3 1 3 1 1 1 2 3 2 2 1 2 3 2 1 2 2 2 1 3 2 1 3 2 2 1 2 3 2 3 1 3 1 1 2 3 2 3 2 2 2 3 1 2 2 2 1 1 3 2 1 2 3 2 2 2 3 2 2 2 1 2 1 3 1 1 2 3 2 1 2 3 3 1 3 2 1 2 1 2 1 3 1 1 3 1 1 1 3 1 1 1 2 2 2 3 1 2 3 1 3 2 3 1 1 3 2 1 1 1 2 3 2 1 3 2 2 1 2 2 2 2 1 1 3 1 1 3 2 3 1 3 2 2 1 2 2 3 2 3 1 2 1 2 1 2 3 1 1 1 2 3 1 3 1 1 2 1 2 2 3 2 2 3 2 2 2 3 3 1 2 2 1 1 2 3 1 2 2 1 2 3 2 3 1 1 2 2 3 1 2 3 3 1 1 1 2 3 2 2 1 1 1 3 1 2 1 2 3 1 1 1 3 2 1 3 2 1 2 2 3 2 2 3 1 2 2 2 3 1 2 1 2 2 1 3 2 3 2 3 2 2 2 1 2 3 2 2 2 3 2 3 2 1 2 3 2 1 1 3 2 1 3 2 1 1 2 2 3 1 1 1 3 1 1 2 2 3 2 3 2 3 1 1 2 2 3 1 2 3 1 3 2 2 2 3 1 1 2 2 2 3 2 2 2 3 1 3 2 1 1 2 3 1 2 3 2 1 2 1 1 2 3 1 2 3 2 3 2 3 2 1 1 1 2 2 1 2 3 2 3 1 3 1 3 1 1 3 1 1 2 2 2 3 2 2 2 1 2 2 3 2 3 1 2 1 1 1 3 2 1 2 2 3 2 2 3 1 2 1 3 1 1 1 3 1 1 3 2 1 3 1 1 2 1 3 1 1 1 3 2 2 1 1 2 1 3 1 2 2 3 2 3 2 1 3 2 2 1 1 3 1 3 2 2 3 2 2 2 1 1 2 2 1 3 2 1 3 2 1 1 3 2 2 3 2 2 1 3 1 1 2 1 3 2 2 1 1 2 2 2 3 1 1 3 2 1 2 1 1 2 3 1 1 2 3 2 3 2 3 2 1 3 1 1 1 2 2 3 2 1 3 2 1 2 2 2 3 1 3 1 3 1 1 2 3 2 1 2 1 2 3 2 2 1 1 2 3 1 3 1 2 3 2 2 3 2 1 2 1 2 2 2 3 1 2 1 1 3 1 3 1 1 2 3 1 1 3 1 1 3 2 2 2 3 1 1 2 1 3 2 3 2 1 1 2 3 1 1 2 1 2 3 1 2 3 3 2 1 3 2 2 2 3 2 3 1 1 2 1 3 1 1 2 2 1 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 2 1 1 2 2 2 3 2 3 2 3 1 1 3 1 1 3 1 2 2 3 2 2 3 1 3 2 2 1 1 2 1 3 1 2 1 1 1 3 1 2 2 1 2 3 2 1 3 2 3 1 2 3 2 1 1 1 2 3 2 2 3 1 1 2 2 2 1 3 1 2 3 2 1 3 1 2 1 2 3 1 1 2 3 2 3 1 2 1 3 1 1 3 2 3 2 1 2 2 1 1 3 2 1 1 3 2 2 1 2 1 2 3 1 1 2 2 1 2 3 1 3 1 1 3 1 1 2 1 3 1 3 2 2 2 2 3 2 2 1 2 3 1 1 3 2 3 1 2 2 2 3 2 2 2 3 2 3 2 1 1 1 3 1 2 2 3 2 3 2 2 1 2 1 2 3 1 1 1 2 3 2 2 3 2 3 1 2 1 3 2 1 3 2 2 1 3 1 2 1 2 2 2 3 2 3 1 1 2 2 1 1 3 1 2 1 1 1 3 1 1 3 1 3 1 1 3 2 1 3 1 2 2 3 2 1 3 1 1 2 3 1 1 2 2 2 3 2 1 3 2 1 2 1 1 1 2 1 1 3 1 3 1 3 1 3 1 1 2 3 1 2 2 2 1 3 2 1 1 2 2 1 2 3 2 3 1 1 2 1 3 1 2 2 3 2 2 3 1 1 3 2 2 1 1 3 1 2 2 2 1 2 3 2 3 1 2 1 3 2 1 3 1 3 2 2 2 1 1 1 3 1 2 1 3 2 3 2 2 2 3 2 2 3 2 3 2 2 1 2 1 2 2 3 1 2 2 2 1 2 3 1 1 3 1 3 2 1 2 1 3 2 3 1 1 1 2 2 2 3 1 2 3 1 3 2 1 3 2 2 2 1 1 3 1 3 1 1 2 1 1 1 3 2 2 3 2 2 2 3 1 2 3 2 2 2 3 1 1 2 3 3 1 2 2 3 2 3 1 2 3 1 1 2 1 1 2 3 2 2 1 2 2 3 1 3 1 2 3 1 1 3 1 1 1 2 1 2 3 1 2 1 2 3 1 1 2 1 3 2 2 1 1 1 3 2 2 1 2 2 3 1 1 3 2 3 1 1 3 2 2 3 1 2 2 3 2 1 1 3 1 1 1 2 1 3 1 3 1 2 2 2 3 2 3 2 2 3 1 3 1 2 2 3 1 3 2 2 2 1 1 3 2 1 2 2 1 3 1 2 2 1 3 2 3 1 2 1 1 2 1 3 1 1 2 3 1 2 1 1 1 2 3 2 3 3 1 2 1 1 2 1 3 2 3 1 1 2 2 2 3 1 3 2 2 3 2 1 2 1 3 1 2 1 2 2 2 3 2 1 3 2 1 3 1 1 1 3 2 1 2 3 2 3 2 2 1 2 3 1 1 2 3 2 2 3 1 1 2 2 2 3 1 1 2 3 2 1 2 3 1 1 1 3 1 2 2 2 1 3 2 2 3 2 3 1 3 1 2 1 2 1 1 1 2 1 3 1 3 1 1 3 2 2 1 2 3 1 2 3 2 3 1 2 1 2 2 1 3 2 3 1 3 1 1 1 2 3 2 2 2 1 1 2 3 2 3 1 2 2 3 1 1 3 1 1 2 1 2 3 2 3 1 1 1 2 2 1 3 2 2 2 3 3 2 2 2 3 1 2 1 3 2 2 2 1 1 2 3 1 3 2 1 2 2 3 1 3 2 2 3 2 1 1 3 2 1 1 2 3 1 2 1 1 1 3 2 1 2 3 1 2 1 1 3 1 3 2 1 3 2 1 1 2 2 3 2 2 3 2 2 2 1 3 1 2 2 2 3 1 3 1 3 1 3 2 1 2 3 2 1 2 3 1 2 2 1 2 2 1 2 2 3 1 2 2 3 2 3 1 1 2 2 1 3 1 2 1 3 1 1 3 1 3 1 2 2 1 3 2 1 2 2 2 1 3 2 1 3 2 1 1 2 1 3 1 3 2 1 2 3 2 1 2 2 1 3 1 3 1 2 1 2 2 3 1 1 1 3 2 3 2 1 2 3 2 3 1 1 1 3 2 1 1 2 3 1 2 1 1 1 2 3 1 3 3 2 1 1 2 2 1 3 2 1 1 2 3 1 2 2 2 3 1 1 2 3 1 3 3 2 2 2 1 2 2 3 2 1 1 1 3 1 2 3 2 1 1 3 2 3 1 1 2 1 3 2 1 3 1 1 2 2 3 2 2 3 2 2 1 1 1 3 1 1 2 3 2 1 2 2 3 2 2 1 3 2 2 1 2 3 2 1 3 2 3 2 3 2 1 1 3 1 3 2 3 1 1 1 3 2 2 1 2 1 2 3 1 1 1 3 2 1 2 1 1 2 1 2 1 3 1 1 3 2 2 3 1 2 3 1 3 2 2 2 1 2 3 1 2 2 2 1 3 1 1 3 2 1 1 3 1 1 2 1 1 3 2 3 1 3 2 1 2 3 2 3 2 1 2 1 1 3 1 2 1 2 2 2 3 2 1 1 3 1 1 3 2 1 3 1 1 3 1 3 2 2 3 2 1 2 2 3 2 2 1 2 1 1 3 2 3 2 3 2 2 1 2 2 1 3 2 2 2 1 1 3 2 2 1 3 1 3 2 1 1 1 2 1 2 1 3 2 3 1 2 3 2 3 1 1 1 2 2 3 1 1 2 3 2 2 1 3 1 3 1 1 2 1 3 1 3 2 3 1 2 2 1 2 1 3 2 2 3 1 1 3 2 3 1 3 2 2 1 1 2 3 1 2 2 2 3 2 1 1 1 2 1 1 2 3 2 1 1 1 3 2 1 1 1 3 1 1 1 3 2 3 1 2 3 1 3 2 2 1 3 2 2 1 2 3 1 2 3 1 1 2 1 2 2 3 2 3 2 1 1 1 1 2 3 1 3 2 2 1 3 1 3 2 1 3 1 1 2 2 1 2 3 2 3 1 2 1 2 1 3 1 1 3 1 2 2 1 3 2 2 1 3 2 3 1 2 1 1 2 1 3 2 2 2 3 2 2 3 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 1 1 2 1 3 2 2 1 3 2 1 3 2 1 1 3 1 3 2 1 2 3 1 1 2 2 2 3 2 1 2 2 3 2 3 1 1 3 2 2 2 1 3 2 1 3 2 1 3 2 1 1 3 1 1 3 1 3 1 1 2 2 1 3 1 2 2 1 1 1 1 2 3 2 3 2 2 1 2 3 2 1 2 3 2 1 1 1 2 1 3 2 3 3 1 1 2 2 1 3 2 2 1 3 1 3 2 1 1 1 2 2 3 2 2 2 3 3 1 1 1 2 2 3 1 1 3 1 2 1 3 2 1 1 3 1 1 1 2 3 1 3 2 3 2 1 2 2 1 2 3 2 3 1 2 2 2 1 2 3 1 2 1 3 1 2 1 2 2 1 2 3 1 3 1 1 1 3 2 2 3 1 1 2 1 3 2 1 3 2 1 2 3 2 1 2 2 3 2 1 2 2 3 1 3 2 1 3 1 2 3 1 1 3 2 3 1 2 2 3 1 1 2 1 3 2 1 3 1 2 2 3 2 2 2 1 1 1 3 2 1 1 3 2 2 3 2 2 2 3 1 2 2 3 1 1 1 2 2 2 3 3 1 1 3 2 2 2 3 1 2 2 2 1 1 3 2 2 2 1 1 3 1 1 3 3 1 3 1 1 3 1 2 1 1 1 2 3 1 2 1 2 2 3 2 2 1 2 3 1 2 3 1 2 3 1 3 2 2 3 2 2 1 1 2 1 3 2 2 1 3 2 2 2 1 2 3 1 2 1 2 2 2 3 1 1 3 1 3 2 3 2 2 1 1 3 1 3 1 3 1 2 3 1 2 2 1 1 1 3 2 3 1 2 2 2 1 2 3 1 1 1 2 1 3 2 2 1 1 3 1 3 2 3 1 2 3 1 3 1 1 2 1 1 1 2 2 2 1 2 2 3 2 2 1 3 1 2 1 1 1 3 1 3 2 2 3 1 3 1 3 1 1 2 1 2 2 3 1 2 1 3 2 2 3 1 1 3 2 2 3 1 1 2 1 3 2 3 2 1 1 1 3 2 3 2 1 3 1 2 2 3 2 1 1 1 2 1 3 2 1 3 2 3 1 2 1 2 3 1 2 2 2 3 1 1 2 1 2 2 3 2 3 2 1 2 2 3 1 1 2 2 1 3 1 1 2 1 3 2 3 1 3 1 1 2 3 1 2 1 2 3 1 3 1 2 1 3 1 1 3 2 2 2 1 1 2 3 2 3 1 1 3 1 1 3 2 1 1 3 2 1 2 1 1 1 3 2 1 1 1 2 3 2 2 2 1 1 3 2 3 2 3 1 2 1 1 3 1 1 1 3 1 2 1 3 1 2 1 2 2 3 2 2 3 1 1 2 3 2 3 2 2 2 1 1 1 3 1 3 1 3 1 1 2 1 1 2 3 1 2 3 1 3 1 2 3 1 2 2 1 2 2 3 1 2 1 3 1 3 1 1 1 3 1 3 1 3 1 1 2 2 3 2 1 2 2 1 1 1 2 3 2 1 2 1 1 2 3 1 3 1 2 1 2 3 2 2 2 3 2 3 1 1 1 2 1 3 1 2 1 1 3 1 2 2 3 1 2 2 3 2 3 2 2 2 3 2 2 2 3 1 2 3 1 2 1 1 2 1 3 1 1 3 1 3 1 1 2 3 1 1 3 1 2 3 1 1 2 1 1 3 2 2 3 2 3 1 1 2 3 2 2 2 1 1 3 1 2 3 1 1 1 3 1 1 1 3 2 3 2 1 3 1 1 2 1 2 2 2 3 2 2 1 1 1 2 3 2 1 2 3 2 1 3 2 1 1 2 2 3 1 3 2 1 3 2 1 3 2 3 2 3 1 1 3 2 2 1 2 2 2 3 2 2 1 2 1 3 2 3 1 1 2 3 2 2 2 3 2 1 1 1 3 1 3 2 2 2 1 1 3 1 2 1 1 1 2 3 1 3 1 1 2 2 3 1 3 2 1 1 2 2 3 2 2 3 1 2 3 1 3 1 1 1 2 2 3 2 2 2 1 1 3 2 3 2 2 2 1 1 1 2 1 1 3 2 1 3 2 3 2 3 1 3 2 1 1 2 1 3 2 1 2 1 2 3 1 1 1 2 1 2 3 2 3 1 2 1 3 2 1 1 3 1 3 1 1 2 2 3 2 1 1 3 1 3 2 3 1 2 2 1 2 1 3 1 2 3 1 2 1 3 1 3 2 1 1 3 1 1 2 3 1 1 1 3 1 3 1 2 1 1 2 1 2 1 1 3 2 1 1 3 2 1 3 1 2 3 2 2 1 1 1 3 1 3 1 2 1 1 1 2 1 3 1 1 1 3 1 1 2 2 3 2 1 3 1 3 2 1 3 2 1 2 1 3 1 2 2 2 1 1 3 2 3 1 1 3 1 3 1 3 2 2 1 2 3 1 1 2 3 2 2 2 3 2 1 1 1 2 3 2 1 2 1 3 1 2 1 3 1 1 1 2 1 3 1 1 2 3 1 3 2 1 3 2 3 1 1 1 2 1 2 3 2 2 3 1 1 2 2 1 2 3 2 1 3 1 3 1 1 1 3 2 1 1 1 3 2 1 3 2 1 1 1 2 2 3 1 3 1 3 2 1 3 2 2 3 1 1 2 2 2 3 2 1 1 1 3 2 3 2 2 2 1 2 1 3 2 3 2 3 2 1 1 2 1 2 1 2 3 1 2 2 2 3 1 3 1 2 3 1 3 1 1 2 3 2 1 1 1 1 2 1 2 2 3 1 2 1 2 3 2 3 2 2 3 2 3 1 1 3 2 1 1 3 2 3 1 3 1 2 2 1 2 3 1 3 2 1 2 2 3 1 2 2 2 1 2 2 3 2 1 2 2 2 1 3 1 2 1 3 2 3 1 3 1 2 2 1 2 3 1 2 1 3 1 1 1 2 3 1 1 1 3 1 2 1 3 1 2 1 3 1 1 3 3 1 2 2 3 2 1 2 1 2 3 2 1 1 1 3 2 1 3 2 2 2 1 3 2 1 2 3 1 1 2 3 2 2 1 2 2 3 2 3 2 3 2 2 3 1 2 2 3 1 2 1 2 2 1 3 2 1 3 1 3 2 1 1 3 2 1 2 1 2 2 3 2 3 1 3 1 2 3 1 1 2 2 2 3 2 3 2 2 1 2 3 1 2 1 2 2 1 2 3 1 1 2 3 1 1 3 2 1 1 1 3 1 3 1 2 3 2 1 1 3 1 3 2 3 1 1 2 2 2 3 2 2 3 2 1 1 2 2 2 3 2 2 2 1 3 1 1 1 2 2 3 2 1 3 1 3 2 2 1 1 2 2 3 2 3 2 1 3 2 3 2 2 1 1 2 3 1 1 1 3 2 2 3 2 3 1 1 2 1 1 2 2 3 2 3 1 2 2 2 3 2 2 1 1 3 1 1 3 1 2 2 1 1 2 3 1 3 2 1 3 2 1 2 2 3 2 1 1 1 3 2 1 2 1 1 1 3 1 3 2 3 1 2 2 3 2 2 3 2 1 2 1 3 2 2 1 2 2 3 2 3 2 1 3 1 2 2 3 2 1 3 2 2 2 1 1 2 3 2 2 1 1 3 1 1 2 3 1 2 3 1 1 1 2 1 1 3 1 1 1 2 2 3 1 3 2 1 3 1 3 1 2 1 2 3 1 2 3 1 2 1 2 2 2 3 2 2 3 2 1 2 3 2 3 2 2 2 2 1 3 1 3 2 2 2 3 1 2 2 1 3 2 1 2 3 2 2 2 3 1 1 2 1 1 3 1 3 1 2 2 3 2 3 1 2 3 1 3 1 1 1 2 1 1 1 2 3 1 1 2 1 3 1 1 2 1 3 1 3 1 1 2 3 2 1 3 1 3 2 1 3 2 1 3 2 1 1 2 2 2 3 1 1 2 3 2 2 2 3 1 1 1 3 2 3 1 3 2 1 1 2 2 3 1 2 2 3 1 2 2 3 2 2 1 1 3 1 1 2 1 1 2 3 2 2 2 1 3 2 3 2 3 2 2 2 3 1 1 1 1 2 1 2 3 1 1 1 3 2 1 3 1 3 1 1 1 3 2 3 2 2 1 2 2 3 1 3 2 2 1 2 2 3 2 1 2 2 2 1 3 2 2 2 3 1 1 3 2 1 3 2 2 3 1 3 2 2 2 1 1 1 3 2 2 3 1 1 1 3 1 1 2 1 1 1 3 1 3 2 3 1 2 3 2 1 1 1 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1 3 1 2 3 1 3 2 2 1 2 3 2 3 2 1 2 3 1 1 3 1 2 2 1 2 1 3 2 2 1 3 3 1 1 1 2 2 3 2 2 3 2 1 2 1 3 1 3 2 3 1 2 1 2 1 2 1 3 1 1 1 2 1 3 2 2 2 1 1 3 2 1 2 1 3 2 3 2 3 2 3 1 2 2 2 1 3 1 2 3 2 2 2 1 2 2 3 2 3 1 3 1 1 1 1 3 2 2 3 1 2 1 2 2 2 3 2 2 3 2 2 1 3 1 2 3 1 2 3 1 2 3 2 3 1 2 1 1 2 3 1 3 1 1 2 1 1 1 3 1 1 1 1 1 3 2 2 2 1 3 2 2 2 3 2 1 2 2 1 3 2 1 3 1 3 1 3 2 2 2 3 1 2 3 2 3 1 2 1 3 2 1 1 1 2 1 3 1 1 1 1 3 2 3 2 2 1 2 2 3 1 1 2 3 2 3 1 2 3 2 2 1 2 1 1 1 2 2 3 1 1 3 2 3 2 3 1 2 1 1 2 3 2 2 2 3 2 3 2 2 2 1 3 2 3 1 2 2 1 1 1 3 1 2 1 3 1 2 2 1 3 1 2 1 1 3 2 3 2 1 2 1 1 3 1 3 1 1 3 2 3 2 2 1 1 1 2 3 1 1 2 2 2 3 2 2 2 3 2 3 1 2 3 1 1 3 2 2 1 1 1 1 3 1 1 2 2 3 1 3 1 1 1 2 3 1 1 1 3 2 2 1 3 1 3 2 3 2 1 1 3 1 3 2 1 2 1 1 1 3 2 1 2 2 2 3 1 3 1 1 2 2 1 1 3 1 2 2 3 2 2 1 2 1 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 2 3 2 2 3 2 2 2 1 1 3 2 1 1 3 1 2 1 1 1 3 2 1 1 1 2 3 2 2 1 2 3 2 3 1 3 1 3 1 1 1 1 1 3 1 2 1 2 2 3 1 2 2 3 1 3 1 2 1 3 1 3 2 2 1 1 3 2 3 1 2 1 2 3 1 1 2 1 2 3 2 3 1 3 1 1 1 2 1 1 1 2 1 3 1 3 2 2 2 3 2 2 1 1 2 3 2 1 1 3 2 3 3 1 2 2 2 1 3 1 2 3 1 3 2 2 1 1 3 1 1 2 2 2 1 3 2 2 3 2 1 1 1 2 3 1 3 2 3 2 3 1 1 2 1 2 2 3 2 1 1 3 2 1 3 2 3 2 1 2 2 2 3 1 3 1 2 1 1 2 1 3 1 1 2 3 1 3 2 2 1 1 1 3 1 3 2 2 3 2 2 3 1 2 1 2 2 2 1 1 1 3 1 3 2 3 2 1 2 2 1 3 1 1 1 2 1 3 2 2 2 3 3 2 2 2 1 3 2 2 1 2 2 2 3 1 2 3 1 3 1 2 1 1 2 3 1 1 3 2 3 2 1 1 1 2 3 1 1 2 1 1 1 3 1 3 2 2 3 2 1 1 2 1 1 1 3 2 3 1 3 2 1 3 1 1 3 2 3 2 1 1 2 2 2 1 2 2 3 1 3 2 2 2 3 2 3 2 1 1 1 3 1 1 3 1 2 1 2 2 2 1 2 1 3 2 2 3 2 2 3 2 3 2 2 3 1 1 1 3 2 2 1 2 3 1 1 1 2 1 2 3 1 2 2 3 2 3 2 2 2 3 2 2 3 2 1 1 1 3 1 3 1 2 3 2 1 1 1 3 2 3 1 3 2 2 1 2 2 1 2 2 3 1 1 3 1 1 1 3 2 2 1 3 1 2 3 1 2 3 1 1 2 2 1 2 3 2 2 1 2 2 2 3 2 2 2 1 3 2 2 2 3 2 3 2 3 1 1 1 1 2 1 2 3 1 1 2 2 2 3 1 1 3 1 3 1 1 3 2 3 1 3 1 2 2 1 3 1 2 1 2 1 3 1 1 2 1 2 2 3 1 1 3 1 3 2 2 1 3 1 1 2 1 1 3 1 3 1 1 1 2 3 2 1 2 3 2 3 2 2 2 2 1 2 3 1 1 1 3 1 3 1 1 3 2 3 2 1 2 2 1 2 3 3 2 1 1 3 2 1 2 2 1 1 3 2 3 2 3 1 2 2 2 1 3 2 1 1 2 1 1 1 3 1 3 1 1 3 2 1 1 1 3 1 3 2 1 1 1 3 2 1 2 2 3 2 2 1 1 2 2 3 1 1 3 2 3 2 1 2 3 1 1 1 3 2 1 2 1 2 1 3 2 2 3 1 3 2 2 3 1 3 2 1 1 3 1 2 2 2 1 3 1 2 3 1 3 1 2 1 2 1 2 3 1 1 1 3 1 2 1 3 2 1 2 1 1 3 1 1 3 1 2 3 1 2 2 2 3 2 3 2 1 1 1 2 3 2 2 1 3 2 1 1 2 1 1 3 1 1 1 3 1 2 3 1 1 3 1 3 1 3 1 2 2 2 3 2 3 1 3 2 1 1 1 3 2 1 1 1 2 1 3 1 1 1 1 1 2 1 3 1 2 3 2 1 3 1 1 2 2 2 3 2 3 2 3 2 2 3 1 1 1 2 2 1 3 2 3 2 2 2 3 2 3 2 3 2 1 2 2 1 2 1 2 2 2 3 1 3 2 1 2 3 1 2 1 3 1 1 3 1 2 2 3 2 2 1 2 1 3 1 3 2 2 3 1 1 3 2 1 2 3 2 1 1 1 3 2 2 2 2 1 1 2 2 2 3 2 3 1 1 2 3 2 2 3 2 2 1 2 2 3 2 3 2 2 1 3 2 2 2 1 2 3 1 3 1 3 2 3 1 3 1 2 2 2 1 1 3 2 2 3 2 2 1 3 1 3 2 3 2 2 2 1 2 3 1 1 1 2 2 2 2 2 2 1 2 2 3 2 3 1 2 3 2 3 1 1 1 2 1 1 3 1 3 1 3 2 1 1 3 2 1 1 2 1 2 3 1 2 1 3 2 3 1 2 2 1 1 3 2 3 1 3 1 2 3 1 2 3 2 1 2 1 2 3 2 1 3 2 1 1 2 1 1 1 2 2 3 1 3 1 1 1 3 1 3 1 2 1 1 1 2 3 1 2 1 3 2 2 2 3 1 1 3 2 3 1 2 3 2 2 1 3 1 1 2 3 2 2 2 1 1 3 2 2 3 2 2 3 2 3 2 1 1 2 2 3 2 2 1 3 2 1 1 1 1 2 1 3 2 3 1 3 1 1 3 2 3 1 2 1 1 3 1 2 1 2 2 2 3 2 3 2 3 1 2 1 1 3 2 1 1 2 2 3 1 3 2 2 1 1 1 2 2 1 3 2 2 1 2 2 3 2 2 2 3 2 3 1 1 2 2 2 3 1 3 1 1 2 1 3 2 2 3 1 1 2 1 3 2 1 1 2 2 2 3 1 1 3 1 3 1 2 3 2 2 2 3 2 3 2 2 2 3 1 1 2 1 3 1 3 1 1 2 1 2 3 1 2 1 1 1 3 1 2 1 2 3 1 3 1 3 1 2 2 3 2 1 1 2 1 1 1 3 1 2 3 1 3 1 2 3 2 2 3 2 2 1 1 1 3 2 2 1 1 3 2 3 1 1 1 2 2 2 3 2 1 1 3 1 1 2 2 1 3 2 3 3 1 1 1 2 3 1 3 1 3 2 2 1 2 2 3 1 2 1 3 2 2 2 1 2 2 2 3 2 1 1 1 2 3 1 3 1 2 1 2 1 3 2 3 2 2 1 3 3 2 2 1 1 2 2 3 2 3 1 2 1 2 2 2 3 1 2 2 1 3 2 3 1 3 1 3 2 3 2 2 3 1 2 1 1 1 3 1 2 3 2 2 2 1 2 1 1 1 2 2 3 2 3 1 3 1 1 1 2 2 3 1 2 1 1 3 1 1 3 1 2 2 1 1 1 3 1 3 1 1 2 2 3 1 3 1 1 3 1 3 1 1 1 2 2 2 3 2 2 1 3 1 1 3 1 1 2 2 3 1 1 2 3 2 1 2 3 2 1 3 2 2 1 1 3 1 2 1 2 3 2 3 2 3 1 2 3 2 2 2 1 1 2 3 1 3 2 2 1 2 3 2 2 3 2 1 1 2 1 3 1 1 1 2 2 3 2 2 1 3 1 2 1 1 3 2 2 2 1 3 1 3 1 2 2 3 1 3 1 1 1 2 3 1 3 2 1 1 2 1 1 3 1 3 2 1 2 2 2 3 1 1 3 2 2 3 2 2 2 1 1 3 2 3 2 1 1 2 3 1 2 2 2 3 2 2 1 3 2 2 3 1 1 3 1 1 3 1 2 2 3 2 2 1 2 2 3 2 2 3 1 1 2 1 2 1 3 1 1 1 3 1 2 2 1 1 1 3 1 3 2 3 1 1 2 3 2 1 1 1 2 2 3 2 2 1 3 1 1 1 2 2 2 3 1 3 2 3 2 3 1 2 2 3 2 2 1 3 2 3 2 3 2 2 1 2 2 3 1 2 2 1 2 3 3 1 3 1 1 2 2 1 2 3 2 3 2 3 1 1 2 1 2 1 3 1 1 1 2 2 3 1 2 2 3 1 2 1 1 1 3 2 1 1 1 3 1 3 2 3 2 1 3 2 3 2 3 2 1 1 1 2 2 3 1 1 2 1 2 3 2 2 1 1 2 3 1 1 1 3 2 1 1 1 3 1 1 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 2 1 3 2 2 3 1 1 3 1 1 2 1 3 1 1 1 3 1 1 1 3 3 2 3 2 1 1 2 1 1 3 1 3 2 3 1 1 2 1 3 2 1 1 2 2 2 1 2 2 3 1 1 1 2 1 1 3 1 3 1 3 1 2 2 2 3 2 3 1 1 2 3 1 3 1 1 1 3 1 1 3 1 1 3 2 2 1 1 3 1 2 2 2 1 1 3 1 3 2 3 1 3 2 1 2 1 2 2 3 2 2 1 1 1 3 1 1 2 2 2 3 2 1 1 1 3 2 3 1 2 3 1 2 3 2 1 1 3 1 2 1 3 1 2 3 2 2 1 2 3 2 3 1 2 3 1 1 1 2 1 2 3 2 1 2 3 2 1 3 1 1 2 1 1 1 3 2 3 2 2 1 1 1 3 2 3 2 2 1 1 1 1 3 1 3 2 1 2 3 2 3 2 3 2 1 2 3 1 2 1 2 2 2 1 1 1 3 1 2 1 1 3 1 3 2 2 1 3 2 1 1 1 2 2 3 2 3 1 1 3 1 1 2 2 1 3 1 3 1 1 2 1 1 3 2 3 2 3 1 2 1 3 1 2 1 1 3 1 1 1 3 2 3 1 1 1 2 3 2 1 1 1 2 2 3 3 1 2 3 1 1 1 3 1 2 3 2 2 2 1 1 1 3 2 2 2 3 2 2 1 3 2 3 2 1 1 3 2 1 1 2 1 1 3 2 2 2 3 1 3 1 1 1 3 2 2 3 1 3 1 1 2 2 1 3 1 1 2 2 2 3 1 2 1 1 1 3 2 2 1 1 3 1 1 1 2 2 2 3 2 1 2 3 2 3 2 2 3 2 2 3 1 3 1 1 3 1 2 2 2 1 3 1 2 3 1 1 1 2 3 1 3 2 2 2 2 1 1 3 2 2 2 3 1 3 1 2 1 1 1 3 1 2 3 1 2 1 2 3 2 3 1 3 1 2 1 3 2 2 2 3 2 1 1 2 1 2 3 2 2 2 3 2 1 3 2 2 2 3 1 1 1 2 2 3 2 1 1 3 2 2 2 3 1 2 3 1 2 1 3 1 1 2 2 3 1 2 2 1 1 2 3 1 2 3 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 2 1 1 2 1 1 3 2 1 2 2 3 1 1 3 1 2 1 1 2 3 1 2 3 2 1 1 2 3 2 1 1 3 2 1 3 2 3 2 2 3 1 1 1 2 2 2 3 1 2 3 1 3 1 3 1 2 1 2 3 2 2 1 2 3 2 3 2 1 1 1 3 2 1 2 1 3 2 2 2 1 2 3 2 2 1 3 2 3 1 1 2 1 1 3 2 3 1 1 1 2 1 3 1 1 2 3 1 1 2 3 1 1 1 3 1 1 1 3 1 2 2 3 2 1 1 2 1 1 3 2 1 3 1 3 1 1 2 3 1 1 1 2 1 3 2 3 2 2 1 1 1 2 3 1 3 2 3 2 3 2 1 3 1 2 1 1 1 3 1 2 3 2 3 1 1 2 2 1 2 3 1 2 3 1 2 1 3 2 1 2 1 2 3 2 3 2 3 2 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 2 1 3 1 1 1 2 3 2 2 2 3 1 1 3 1 2 1 1 1 2 2 2 3 2 1 3 1 3 1 3 1 1 1 2 2 2 3 2 2 3 2 1 3 1 1 2 1 1 3 1 2 2 1 3 2 1 1 3 2 3 2 1 3 1 2 3 1 2 2 2 1 3 1 3 1 1 1 2 1 2 3 1 3 2 1 3 1 1 1 1 2 1 3 1 3 2 1 2 3 2 2 3 2 2 2 1 2 3 1 3 1 1 3 1 2 3 1 2 3 1 1 3 1 3 2 2 2 1 2 2 3 2 1 1 1 2 1 1 3 2 1 1 1 3 1 1 3 1 1 3 1 1 1 2 3 2 3 2 2 1 2 2 3 1 1 3 1 1 2 2 1 1 3 2 3 2 2 2 1 3 2 3 2 1 1 3 1 1 1 3 1 1 2 3 2 2 3 1 2 2 2 1 2 3 1 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 2 3 1 2 2 3 1 1 1 2 1 1 2 3 1 2 1 1 2 2 3 2 2 3 1 3 1 3 1 3 2 1 1 2 3 2 2 2 3 2 2 3 1 1 1 3 2 3 2 1 1 1 3 2 1 2 1 2 2 3 1 3 2 2 1 2 1 2 3 1 3 1 1 1 3 2 3 2 1 1 2 2 2 2 3 2 3 1 3 1 1 1 3 1 1 3 2 1 2 1 2 1 3 1 1 2 3 2 1 1 3 2 2 2 1 3 1 3 2 2 1 2 1 3 1 3 2 2 2 1 3 1 2 1 3 1 2 1 3 1 2 1 1 3 2 2 1 1 2 2 3 1 1 3 1 3 1 3 1 2 3 1 2 2 3 2 2 2 1 2 3 2 1 2 2 1 2 3 1 1 1 3 2 2 1 1 3 1 1 1 2 2 3 2 1 3 2 3 1 2 1 3 2 2 2 3 1 2 1 2 2 3 2 2 2 3 2 3 1 3 2 3 2 1 2 1 1 2 2 2 3 2 1 3 1 1 1 3 2 2 3 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 2 1 1 3 2 2 2 1 2 1 3 1 2 3 1 1 1 1 3 1 2 1 1 1 3 2 3 1 3 2 2 3 1 2 2 2 1 3 1 2 3 1 2 1 2 2 3 2 1 1 3 1 2 1 2 3 2 2 3 2 1 1 1 3 3 2 1 1 3 1 3 2 3 2 1 2 2 3 2 1 1 3 2 2 1 1 2 2 3 2 3 2 3 1 2 2 1 3 2 1 1 2 3 1 1 3 2 1 2 2 2 1 3 2 1 1 3 1 1 1 3 1 2 2 1 1 3 2 3 2 2 1 3 2 1 1 1 3 2 1 3 1 1 1 3 2 2 3 1 1 1 2 2 3 1 2 2 1 2 3 2 1 1 3 1 3 1 1 3 2 2 3 1 3 2 1 1 2 3 2 1 2 2 2 3 2 2 1 1 3 1 1 1 2 1 3 2 1 3 1 2 1 1 3 2 3 1 1 2 1 1 3 2 1 1 1 2 2 3 1 1 1 3 2 3 2 1 2 1 3 2 3 1 1 3 1 2 3 2 1 2 3 2 2 2 1 2 2 3 2 2 3 2 3 2 1 1 2 2 2 1 3 1 1 2 1 2 1 3 2 3 1 1 3 1 3 1 2 1 3 3 2 2 1 2 3 1 1 1 3 1 3 2 1 2 3 2 3 2 2 1 1 1 2 2 1 2 2 1 2 3 2 3 1 1 3 1 1 3 1 1 2 3 1 2 2 1 3 2 1 1 2 1 1 3 2 2 3 1 1 3 1 3 1 1 2 2 3 2 2 3 2 2 3 1 2 3 2 2 2 3 1 2 3 2 1 1 2 2 3 2 2 1 1 1 3 3 2 3 1 1 1 3 1 2 2 2 3 1 3 2 2 2 3 2 1 2 1 1 2 1 3 1 3 1 1 2 1 2 1 3 1 2 2 3 1 3 1 2 2 2 3 2 2 2 2 2 3 1 3 1 2 3 2 3 1 2 3 1 2 1 1 1 3 2 2 1 1 3 2 2 3 2 1 1 1 2 2 3 2 1 3 2 1 1 1 3 1 1 3 2 1 3 2 3 2 2 1 2 3 1 2 3 2 2 3 2 2 2 3 2 1 2 2 1 2 1 2 2 1 2 2 3 2 3 2 1 3 1 2 3 2 1 2 2 1 1 3 1 3 3 2 2 1 3 1 1 1 3 1 2 2 2 1 3 1 1 3 2 2 1 3 2 2 2 2 3 2 3 2 1 2 2 1 1 3 1 3 1 3 2 3 1 1 1 2 1 2 3 2 2 2 1 1 3 1 2 1 3 1 1 1 3 1 3 2 3 1 2 2 2 1 1 1 2 3 1 3 1 1 1 2 1 3 1 2 1 3 2 2 1 2 2 3 2 3 2 3 1 1 2 2 3 1 1 2 1 1 3 1 1 2 2 2 3 2 2 3 2 3 1 1 2 1 1 3 1 2 2 3 1 1 2 2 1 3 2 3 1 3 2 1 1 3 1 1 2 3 2 2 2 3 1 3 1 3 1 2 2 2 1 3 2 1 1 1 3 1 1 3 2 2 2 1 3 1 1 2 1 3 1 1 1 2 3 2 3 2 2 2 3 1 2 1 2 1 1 3 2 1 1 3 2 3 2 2 1 1 3 1 2 2 2 3 1 3 3 2 1 3 2 3 1 1 2 1 1 3 2 2 1 3 2 3 2 2 1 1 2 1 1 1 3 2 3 2 3 2 2 1 1 1 3 2 1 1 1 2 3 2 1 3 1 2 1 3 1 3 1 2 3 2 2 2 1 2 3 2 2 3 2 3 1 1 2 2 1 1 1 3 2 2 3 1 1 2 1 2 2 3 1 2 3 1 2 1 1 3 1 1 3 1 2 3 1 1 2 3 2 3 1 3 1 2 3 2 2 2 1 3 1 1 2 1 1 2 1 1 2 1 1 2 3 1 2 3 2 1 1 3 2 2 2 3 1 3 2 2 2 3 1 1 1 3 1 3 2 3 1 1 2 1 3 1 1 1 2 1 1 3 1 3 1 1 1 1 2 1 1 1 3 2 2 1 2 2 3 1 3 1 3 1 3 2 2 2 1 3 1 3 2 1 3 2 3 2 2 3 2 1 3 2 2 2 1 3 2 1 2 1 2 1 3 2 1 1 3 1 1 2 3 2 1 2 2 1 3 1 2 1 2 2 2 3 2 3 3 1 2 1 1 1 2 3 2 2 2 3 1 2 1 1 1 3 2 1 3 2 2 3 1 2 1 3 2 1 2 3 2 1 2 3 2 3 2 3 1 1 3 1 2 2 2 1 1 2 3 1 1 2 3 2 1 3 1 3 2 3 1 2 2 1 3 2 2 2 1 1 3 2 1 3 2 1 2 2 2 1 3 2 3 1 2 3 2 1 1 3 1 1 2 1 1 3 1 1 2 2 3 2 1 2 2 3 1 1 3 1 1 3 1 1 2 1 2 3 2 2 2 1 2 1 3 1 1 2 2 3 1 3 1 3 1 1 3 2 2 1 1 3 1 1 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 1 3 1 1 2 2 1 2 2 2 1 1 1 3 1 1 1 3 2 1 2 2 3 2 1 1 3 1 3 2 3 1 1 1 2 2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 1 3 2 2 2 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1 2 2 2 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1 1 3 1 3 1 1 2 1 1 2 3 2 1 3 1 3 1 2 1 2 1 1 3 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 1 1 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1 2 2 3 2 3 2 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 3 1 2 2 3 1 2 3 2 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 1 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 2 2 1 1 1 2 3 1 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 1 2 3 1 1 2 1 1 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 2 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 2 2 2 3 1 1 2 3 1 1 1 2 2 3 1 2 3 1 2 1 3 1 2 3 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 3 1 1 1 3 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 2 2 2 1 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 2 1 1 1 3 2 3 1 1 1 3 1 2 2 2 3 1 1 1 2 3 1 2 3 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 2 2 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 2 2 3 2 3 1 1 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 2 2 3 2 3 2 2 3 1 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 2 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 2 2 3 2 1 2 2 2 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 1 3 1 1 1 2 1 2 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 2 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 3 2 2 3 2 1 1 2 2 2 3 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 2 2 3 2 3 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1 3 2 3 1 3 1 2 2 2 1 3 1 1 3 1 2 3 2 2 1 2 2 1 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 3 2 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2

for any pair of sequences of the set:

M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19, where:

M1 is the maximum number of matches for any alignment in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment;

M3 is the maximum number of matches for any alignment having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein:

the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:

for each of (i) to (iv):

(i) m=6, mm=6, og=0 and eg=6,

(ii) m=6, mm=6, og=5 and eg=1,

(iii) m=6, mm=0.2, og=5 and eg=1, and

(iv) m=6, mm=6, og=6 and eg=0,

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and

wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv).

c) detecting said non-target cleavage products.

45. The method of claim 44, wherein said reaction temperature is between approximately 50 and 70 degrees centigrade.

46. The method of claim 44; wherein said target nucleic acid molecules comprises single-stranded DNA.

47. The method of claim 44, wherein said target nucleic acid molecules comprises double-stranded DNA and prior to step (c), said reaction mixture is treated such that said double-stranded DNA is rendered substantially single-stranded.

48. The method of claim 47, wherein said treatment to render said double-stranded DNA is rendered substantially single-stranded by increasing the temperature.

49. The method of claim 44, wherein said target nucleic acid molecules comprises RNA and wherein said first and second oligonucleotide molecules comprise DNA.

50. The method of claim 44, wherein said cleavage means comprises a thermostable 5′ nuclease.

51. The method of claim 50, wherein a portion of the amino acid sequence is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a thermophilic organism.

52. The method of claim 51, wherein said organism is selected from the group consisting of Thermus aquaticus, Thermus flavus and Thermus thermophilus.

53. The method of claim 44, wherein said source of target nucleic acid molecules comprises a sample containing genomic DNA.

54. The method of claim 44, wherein said reaction conditions comprise providing a source of divalent cations.

55. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products, the method comprising:

a) providing:

i) a cleavage means,

ii) a first target nucleic acid, said first target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region;

wherein:

(B) each of 1 to 22 is selected so as to be different from all of the others of 1 to 22;

(B) each of W, X and Y is a base in which:

(i) (a) W=one of A, T/U, G, and C,

X=one of A, T/U, G, and C,

Y=one of A, T/U, G, and C,

and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,

(b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y, or

(ii) (a) W=G or C,

X=A or T/U,

Y=A or T/U,

and X≠Y, and

(b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in all the sequences;

(D) up to three bases can be inserted at any location of any of the sequences or up to three bases can be deleted from any of the sequences;

(E) all of the sequences of a said group of oligonucleotides are read 5′ to 3′ or are read 3′ to 5′; and

wherein each oligonucleotide of a said set has a sequence of at least ten contiguous bases of the sequence on which it is based, provided that:

(iii) the phantom sequence is not greater than or equal to ( 11/12×L) in length;

where L=L₁, or if L₁≠L₂, where L is the greater of L₁and L₂; and

wherein any base present may be substituted by an analogue thereof;

v) a second target nucleic acid, distinct from said first target nucleic acid, and having a fourth region, a fifth region and a sixth region, wherein said fourth region is located adjacent to and downstream from said fifth region, and said fifth region is located adjacent to and downstream from said sixth region, said fifth region having a sequence complementary to said 3′ portion of said sequence selected from the group of sequences listed in step (a)(iv), said sixth region having a sequence complementary to said 5′ portion of the sequence selected from the group of sequences in step (a)(iv);

vi) a third oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said third oligonucleotide having a sequence complementary to said fourth region of said second target nucleic acid, said central portion of said third oligonucleotide having a sequence complementary to said fifth region of said second target nucleic acid, and said 3′ portion of said third oligonucleotide having a sequence that is not base paired to either said second target nucleic acid or said first target nucleic acid and is selected from a set of oligonucleotides based on the group of sequences listed in step (a)(iv) such that said sequence selected is distinct from said sequence selected in step (a)(iv);

b) mixing said cleavage means, said first target nucleic acid, said second target nucleic acid, said first, second, and third oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said first target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said first target nucleic acid so as to create a first cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said first target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, wherein cleavage of said first cleavage structure occurs to generate a first non-target cleavage product, and wherein at least said 5′ portion first non-target cleavage product is annealed to said second target nucleic acid and at least said 5′ and central portion of said third oligonucleotide is annealed to said second target nucleic acid so as to create a second cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said non-target cleavage product when annealed to said second target nucleic acid is greater than the melting temperature of said 5′ and central portion of said third oligonucleotide, wherein cleavage of said second cleavage structure occurs to generate a second non-target cleavage product; and

c) detecting said second non-target cleavage product.

56. The method of claim 55, wherein said first target nucleic acid is genomic DNA and said second target nucleic acid is synthetic DNA.

57. The method of claim 55, wherein said synthetic DNA has at least one hairpin loop.

58. The method of claim 57, wherein the method includes a plurality of said first target nucleic acid sequences, a plurality of first oligonucleotide molecules, a plurality of said second oligonucleotide molecules, a plurality of said second target nucleic acid sequences and a plurality of third oligonucleotide molecules.

59. A method of analyzing a biological sample comprising a plurality of target nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule, the method comprising:

a) providing:

i) a cleavage means,

wherein:

(B) each of 1 to 22 is selected so as to be different from all of the others of 1 to 22;

each of W, X and Y is a base in which:

(i) (a) W=one of A, T/U, G, and C,

X=one of A, T/U, G, and C,

Y=one of A, T/U, G, and C,

and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,

(b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y, or

(ii) (a) W=G or C,

X=A or T/U,

Y=A or T/U,

and X═Y, and

(b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in all the sequences;

(D) up to three bases can be inserted at any location of any of the sequences or up to three bases can be deleted from any of the sequences;

(E) all of the sequences of a said group of oligonucleotides are read 5′ to 3′ or are read 3′ to 5′; and

wherein each oligonucleotide of a said set has a sequence of at least ten contiguous bases of the sequence on which it is based, provided that:

(iii) the phantom sequence is not greater than or equal to ( 11/12×L) in length;

where L=L₁, or if L₁≠L₂, where L is the greater of L₁and L₂; and

wherein any base present may be substituted by an analogue thereof;

c) detecting said second non-target cleavage product.

60. The method of claim 59, wherein said first target nucleic acid is genomic DNA and said second target nucleic acid is synthetic DNA.

61. The method of claim 60, wherein said synthetic DNA has at least one hairpin loop.

62. The method of claim 61, wherein the method includes a plurality of said first target nucleic acid sequences, a plurality of first oligonucleotide molecules, a plurality of said second oligonucleotide molecules, a plurality of said second target nucleic acid sequences and a plurality of third oligonucleotide molecules.

63. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products, the method comprising:

a) providing:

i) a cleavage means,

1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 1 2 3 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 3 2 3 1 3 1 2 1 2 1 3 1 2 2 2 1 3 1 1 1 3 2 1 1 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 2 1 3 2 1 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 1 2 2 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 1 3 1 2 1 1 3 1 1 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 3 2 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 2 2 2 3 2 3 1 1 3 1 2 3 1 1 3 2 1 2 2 2 3 2 1 2 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 2 1 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 3 2 2 1 2 2 2 3 2 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 3 1 1 2 1 3 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 1 2 2 3 2 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 2 3 1 3 1 1 2 3 2 1 1 1 3 1 1 2 3 2 2 2 1 2 2 3 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 3 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 3 1 2 2 3 1 2 1 2 2 2 3 1 1 2 3 2 2 2 3 2 2 2 3 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 2 1 2 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 1 1 1 2 2 2 3 1 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 2 2 3 2 2 3 2 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 1 2 3 2 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 2 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 1 1 3 1 1 1 2 3 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 1 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 1 2 1 3 1 2 3 2 1 1 3 1 3 1 1 1 2 2 3 2 3 1 1 1 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 3 1 3 2 1 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 1 1 2 3 2 1 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 1 2 3 2 2 3 1 3 2 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 3 1 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 2 1 2 2 3 2 1 3 1 1 3 2 1 1 1 3 2 2 1 3 1 1 3 2 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 2 3 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 1 2 1 1 3 1 1 1 2 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 3 1 2 1 2 1 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 3 1 2 2 1 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 2 1 2 3 2 3 2 3 2 2 3 2 2 2 1 3 2 3 2 2 1 2 2 1 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 3 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 2 1 3 2 1 2 2 1 3 2 1 1 3 2 1 2 3 2 3 1 2 2 3 2 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 1 1 2 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 3 1 2 2 3 1 1 2 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 2 1 1 3 2 2 1 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 2 2 2 1 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 3 1 2 1 3 1 2 2 2 1 3 1 1 2 3 1 1 2 2 1 1 3 2 3 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 1 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 2 3 2 2 2 1 2 3 2 1 3 2 3 2 1 3 1 2 2 3 1 1 2 2 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 2 1 3 2 1 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 2 1 3 2 2 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 3 2 1 1 2 3 1 2 1 1 2 3 1 1 3 2 3 2 1 2 1 2 1 3 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 2 1 1 2 3 1 1 3 1 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 2 1 3 2 2 1 3 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 3 1 1 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 1 3 2 2 1 2 1 1 3 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 2 3 2 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 3 1 3 2 2 1 1 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 1 3 2 1 2 1 2 1 3 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 2 1 2 3 2 1 1 1 3 1 1 1 3 2 3 1 1 1 3 1 1 3 1 1 2 3 1 1 2 3 2 1 3 1 1 1 2 3 1 1 2 3 2 2 3 1 1 1 1 1 2 2 3 1 1 2 1 3 2 3 2 3 2 3 1 3 2 2 2 1 1 2 1 3 1 2 1 2 2 3 2 2 2 3 1 2 2 1 1 2 3 1 1 3 1 3 1 1 1 3 2 2 3 2 1 1 1 3 2 2 3 1 1 3 1 2 1 1 1 3 3 2 2 1 1 3 1 3 1 2 2 1 2 3 1 3 1 2 3 2 1 2 2 1 1 3 1 1 3 1 2 1 2 1 1 3 1 1 3 1 2 2 3 1 1 2 2 3 3 2 1 3 1 1 1 2 2 2 3 1 1 2 2 3 1 2 3 2 3 1 1 1 1 1 3 1 3 2 1 3 1 2 2 3 1 2 1 1 3 2 1 2 1 2 3 1 2 3 1 2 1 2 1 3 2 1 3 2 3 1 1 3 1 1 1 2 1 1 3 2 1 3 1 2 1 1 2 3 1 2 3 1 3 1 1 1 2 3 1 1 3 1 2 1 1 2 3 2 3 1 1 1 3 2 1 2 2 2 3 2 3 1 2 1 2 1 3 2 1 1 2 1 1 3 1 3 1 1 2 2 3 1 2 1 2 3 1 1 3 1 2 3 2 1 1 3 2 3 2 1 2 2 2 1 3 2 1 3 1 1 2 3 1 1 3 2 2 1 2 3 2 2 1 3 1 2 2 2 3 2 2 3 1 3 1 2 2 3 1 2 1 3 2 2 2 3 2 1 2 3 1 1 3 1 3 1 2 1 3 2 1 2 2 2 3 1 3 1 1 1 2 3 2 2 1 2 3 2 1 2 2 2 1 3 2 1 3 2 2 1 2 3 2 3 1 3 1 1 2 3 2 3 2 2 2 3 1 2 2 2 1 1 3 2 1 2 3 2 2 2 3 2 2 2 1 2 1 3 1 1 2 3 2 1 2 3 3 1 3 2 1 2 1 2 1 3 1 1 3 1 1 1 3 1 1 1 2 2 2 3 1 2 3 1 3 2 3 1 1 3 2 1 1 1 2 3 2 1 3 2 2 1 2 2 2 2 1 1 3 1 1 3 2 3 1 3 2 2 1 2 2 3 2 3 1 2 1 2 1 2 3 1 1 1 2 3 1 3 1 1 2 1 2 2 3 2 2 3 2 2 2 3 3 1 2 2 1 1 2 3 1 2 2 1 2 3 2 3 1 1 2 2 3 1 2 3 3 1 1 1 2 3 2 2 1 1 1 3 1 2 1 2 3 1 1 1 3 2 1 3 2 1 2 2 3 2 2 3 1 2 2 2 3 1 2 1 2 2 1 3 2 3 2 3 2 2 2 1 2 3 2 2 2 3 2 3 2 1 2 3 2 1 1 3 2 1 3 2 1 1 2 2 3 1 1 1 3 1 1 2 2 3 2 3 2 3 1 1 2 2 3 1 2 3 1 3 2 2 2 3 1 1 2 2 2 3 2 2 2 3 1 3 2 1 1 2 3 1 2 3 2 1 2 1 1 2 3 1 2 3 2 3 2 3 2 1 1 1 2 2 1 2 3 2 3 1 3 1 3 1 1 3 1 1 2 2 2 3 2 2 2 1 2 2 3 2 3 1 2 1 1 1 3 2 1 2 2 3 2 2 3 1 2 1 3 1 1 1 3 1 1 3 2 1 3 1 1 2 1 3 1 1 1 3 2 2 1 1 2 1 3 1 2 2 3 2 3 2 1 3 2 2 1 1 3 1 3 2 2 3 2 2 2 1 1 2 2 1 3 2 1 3 2 1 1 3 2 2 3 2 2 1 3 1 1 2 1 3 2 2 1 1 2 2 2 3 1 1 3 2 1 2 1 1 2 3 1 1 2 3 2 3 2 3 2 1 3 1 1 1 2 2 3 2 1 3 2 1 2 2 2 3 1 3 1 3 1 1 2 3 2 1 2 1 2 3 2 2 1 1 2 3 1 3 1 2 3 2 2 3 2 1 2 1 2 2 2 3 1 2 1 1 3 1 3 1 1 2 3 1 1 3 1 1 3 2 2 2 3 1 1 2 1 3 2 3 2 1 1 2 3 1 1 2 1 2 3 1 2 3 3 2 1 3 2 2 2 3 2 3 1 1 2 1 3 1 1 2 2 1 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 2 1 1 2 2 2 3 2 3 2 3 1 1 3 1 1 3 1 2 2 3 2 2 3 1 3 2 2 1 1 2 1 3 1 2 1 1 1 3 1 2 2 1 2 3 2 1 3 2 3 1 2 3 2 1 1 1 2 3 2 2 3 1 1 2 2 2 1 3 1 2 3 2 1 3 1 2 1 2 3 1 1 2 3 2 3 1 2 1 3 1 1 3 2 3 2 1 2 2 1 1 3 2 1 1 3 2 2 1 2 1 2 3 1 1 2 2 1 2 3 1 3 1 1 3 1 1 2 1 3 1 3 2 2 2 2 3 2 2 1 2 3 1 1 3 2 3 1 2 2 2 3 2 2 2 3 2 3 2 1 1 1 3 1 2 2 3 2 3 2 2 1 2 1 2 3 1 1 1 2 3 2 2 3 2 3 1 2 1 3 2 1 3 2 2 1 3 1 2 1 2 2 2 3 2 3 1 1 2 2 1 1 3 1 2 1 1 1 3 1 1 3 1 3 1 1 3 2 1 3 1 2 2 3 2 1 3 1 1 2 3 1 1 2 2 2 3 2 1 3 2 1 2 1 1 1 2 1 1 3 1 3 1 3 1 3 1 1 2 3 1 2 2 2 1 3 2 1 1 2 2 1 2 3 2 3 1 1 2 1 3 1 2 2 3 2 2 3 1 1 3 2 2 1 1 3 1 2 2 2 1 2 3 2 3 1 2 1 3 2 1 3 1 3 2 2 2 1 1 1 3 1 2 1 3 2 3 2 2 2 3 2 2 3 2 3 2 2 1 2 1 2 2 3 1 2 2 2 1 2 3 1 1 3 1 3 2 1 2 1 3 2 3 1 1 1 2 2 2 3 1 2 3 1 3 2 1 3 2 2 2 1 1 3 1 3 1 1 2 1 1 1 3 2 2 3 2 2 2 3 1 2 3 2 2 2 3 1 1 2 3 3 1 2 2 3 2 3 1 2 3 1 1 2 1 1 2 3 2 2 1 2 2 3 1 3 1 2 3 1 1 3 1 1 1 2 1 2 3 1 2 1 2 3 1 1 2 1 3 2 2 1 1 1 3 2 2 1 2 2 3 1 1 3 2 3 1 1 3 2 2 3 1 2 2 3 2 1 1 3 1 1 1 2 1 3 1 3 1 2 2 2 3 2 3 2 2 3 1 3 1 2 2 3 1 3 2 2 2 1 1 3 2 1 2 2 1 3 1 2 2 1 3 2 3 1 2 1 1 2 1 3 1 1 2 3 1 2 1 1 1 2 3 2 3 3 1 2 1 1 2 1 3 2 3 1 1 2 2 2 3 1 3 2 2 3 2 1 2 1 3 1 2 1 2 2 2 3 2 1 3 2 1 3 1 1 1 3 2 1 2 3 2 3 2 2 1 2 3 1 1 2 3 2 2 3 1 1 2 2 2 3 1 1 2 3 2 1 2 3 1 1 1 3 1 2 2 2 1 3 2 2 3 2 3 1 3 1 2 1 2 1 1 1 2 1 3 1 3 1 1 3 2 2 1 2 3 1 2 3 2 3 1 2 1 2 2 1 3 2 3 1 3 1 1 1 2 3 2 2 2 1 1 2 3 2 3 1 2 2 3 1 1 3 1 1 2 1 2 3 2 3 1 1 1 2 2 1 3 2 2 2 3 3 2 2 2 3 1 2 1 3 2 2 2 1 1 2 3 1 3 2 1 2 2 3 1 3 2 2 3 2 1 1 3 2 1 1 2 3 1 2 1 1 1 3 2 1 2 3 1 2 1 1 3 1 3 2 1 3 2 1 1 2 2 3 2 2 3 2 2 2 1 3 1 2 2 2 3 1 3 1 3 1 3 2 1 2 3 2 1 2 3 1 2 2 1 2 2 1 2 2 3 1 2 2 3 2 3 1 1 2 2 1 3 1 2 1 3 1 1 3 1 3 1 2 2 1 3 2 1 2 2 2 1 3 2 1 3 2 1 1 2 1 3 1 3 2 1 2 3 2 1 2 2 1 3 1 3 1 2 1 2 2 3 1 1 1 3 2 3 2 1 2 3 2 3 1 1 1 3 2 1 1 2 3 1 2 1 1 1 2 3 1 3 3 2 1 1 2 2 1 3 2 1 1 2 3 1 2 2 2 3 1 1 2 3 1 3 3 2 2 2 1 2 2 3 2 1 1 1 3 1 2 3 2 1 1 3 2 3 1 1 2 1 3 2 1 3 1 1 2 2 3 2 2 3 2 2 1 1 1 3 1 1 2 3 2 1 2 2 3 2 2 1 3 2 2 1 2 3 2 1 3 2 3 2 3 2 1 1 3 1 3 2 3 1 1 1 3 2 2 1 2 1 2 3 1 1 1 3 2 1 2 1 1 2 1 2 1 3 1 1 3 2 2 3 1 2 3 1 3 2 2 2 1 2 3 1 2 2 2 1 3 1 1 3 2 1 1 3 1 1 2 1 1 3 2 3 1 3 2 1 2 3 2 3 2 1 2 1 1 3 1 2 1 2 2 2 3 2 1 1 3 1 1 3 2 1 3 1 1 3 1 3 2 2 3 2 1 2 2 3 2 2 1 2 1 1 3 2 3 2 3 2 2 1 2 2 1 3 2 2 2 1 1 3 2 2 1 3 1 3 2 1 1 1 2 1 2 1 3 2 3 1 2 3 2 3 1 1 1 2 2 3 1 1 2 3 2 2 1 3 1 3 1 1 2 1 3 1 3 2 3 1 2 2 1 2 1 3 2 2 3 1 1 3 2 3 1 3 2 2 1 1 2 3 1 2 2 2 3 2 1 1 1 2 1 1 2 3 2 1 1 1 3 2 1 1 1 3 1 1 1 3 2 3 1 2 3 1 3 2 2 1 3 2 2 1 2 3 1 2 3 1 1 2 1 2 2 3 2 3 2 1 1 1 1 2 3 1 3 2 2 1 3 1 3 2 1 3 1 1 2 2 1 2 3 2 3 1 2 1 2 1 3 1 1 3 1 2 2 1 3 2 2 1 3 2 3 1 2 1 1 2 1 3 2 2 2 3 2 2 3 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 1 1 2 1 3 2 2 1 3 2 1 3 2 1 1 3 1 3 2 1 2 3 1 1 2 2 2 3 2 1 2 2 3 2 3 1 1 3 2 2 2 1 3 2 1 3 2 1 3 2 1 1 3 1 1 3 1 3 1 1 2 2 1 3 1 2 2 1 1 1 1 2 3 2 3 2 2 1 2 3 2 1 2 3 2 1 1 1 2 1 3 2 3 3 1 1 2 2 1 3 2 2 1 3 1 3 2 1 1 1 2 2 3 2 2 2 3 3 1 1 1 2 2 3 1 1 3 1 2 1 3 2 1 1 3 1 1 1 2 3 1 3 2 3 2 1 2 2 1 2 3 2 3 1 2 2 2 1 2 3 1 2 1 3 1 2 1 2 2 1 2 3 1 3 1 1 1 3 2 2 3 1 1 2 1 3 2 1 3 2 1 2 3 2 1 2 2 3 2 1 2 2 3 1 3 2 1 3 1 2 3 1 1 3 2 3 1 2 2 3 1 1 2 1 3 2 1 3 1 2 2 3 2 2 2 1 1 1 3 2 1 1 3 2 2 3 2 2 2 3 1 2 2 3 1 1 1 2 2 2 3 3 1 1 3 2 2 2 3 1 2 2 2 1 1 3 2 2 2 1 1 3 1 1 3 3 1 3 1 1 3 1 2 1 1 1 2 3 1 2 1 2 2 3 2 2 1 2 3 1 2 3 1 2 3 1 3 2 2 3 2 2 1 1 2 1 3 2 2 1 3 2 2 2 1 2 3 1 2 1 2 2 2 3 1 1 3 1 3 2 3 2 2 1 1 3 1 3 1 3 1 2 3 1 2 2 1 1 1 3 2 3 1 2 2 2 1 2 3 1 1 1 2 1 3 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2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 1 3 2 2 2 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1 2 2 2 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1 1 3 1 3 1 1 2 1 1 2 3 2 1 3 1 3 1 2 1 2 1 1 3 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 1 1 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1 2 2 3 2 3 2 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 3 1 2 2 3 1 2 3 2 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 1 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 2 2 1 1 1 2 3 1 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 1 2 3 1 1 2 1 1 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 2 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 2 2 2 3 1 1 2 3 1 1 1 2 2 3 1 2 3 1 2 1 3 1 2 3 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 3 1 1 1 3 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 2 2 2 1 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 2 1 1 1 3 2 3 1 1 1 3 1 2 2 2 3 1 1 1 2 3 1 2 3 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 2 2 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 2 2 3 2 3 1 1 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 2 2 3 2 3 2 2 3 1 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 2 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 2 2 3 2 1 2 2 2 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 1 3 1 1 1 2 1 2 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 2 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 3 2 2 3 2 1 1 2 2 2 3 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 2 2 3 2 3 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1 3 2 3 1 3 1 2 2 2 1 3 1 1 3 1 2 3 2 2 1 2 2 1 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 3 2 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2

Wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that:

for any pair of sequences of the set:

M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19, where:

M1 is the maximum number of matches for any alignment in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment;

M3 is the maximum number of matches for any alignment having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein:

the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:

for each of (i) to (iv):

(i) m=6, mm=6, og=0 and eg=6,

(ii) m=6, mm=6, og=5 and eg=1,

(iii) m=6, mm=2, og=5 and eg=1, and

(iv) m=6, mm=6, og=6 and eg=0,

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and

wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv).

c) detecting said second non-target cleavage product.

64. The method of claim 63, wherein said first target nucleic acid is genomic DNA and said second target nucleic acid is synthetic DNA.

65. The method of claim 64, wherein said synthetic DNA has at least one hairpin loop.

66. The method of claim 65, wherein the method includes a plurality of said first target nucleic acid sequences, a plurality of first oligonucleotide molecules, a plurality of said second oligonucleotide molecules, a plurality of said second target nucleic acid sequences and a plurality of third oligonucleotide molecules.

67. A method of analyzing a biological sample comprising a plurality of target nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule, the method comprising:

a) providing:

i) a cleavage means,

iv) a second oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central portion of said second oligonucleotide having a sequence complimentary to said second region of said target nucleic acid, and said 3′ portion of said 20 second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides, based on a following group of sequences each having a 3′ and 5′ portion,

1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 1 2 3 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 3 2 3 1 3 1 2 1 2 1 3 1 2 2 2 1 3 1 1 1 3 2 1 1 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 2 1 3 2 1 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 1 2 2 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 1 3 1 2 1 1 3 1 1 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 3 2 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 2 2 2 3 2 3 1 1 3 1 2 3 1 1 3 2 1 2 2 2 3 2 1 2 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 2 1 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 3 2 2 1 2 2 2 3 2 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 3 1 1 2 1 3 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 1 2 2 3 2 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 2 3 1 3 1 1 2 3 2 1 1 1 3 1 1 2 3 2 2 2 1 2 2 3 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 3 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 3 1 2 2 3 1 2 1 2 2 2 3 1 1 2 3 2 2 2 3 2 2 2 3 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 2 1 2 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 1 1 1 2 2 2 3 1 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 2 2 3 2 2 3 2 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 1 2 3 2 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 2 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 1 1 3 1 1 1 2 3 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 1 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 1 2 1 3 1 2 3 2 1 1 3 1 3 1 1 1 2 2 3 2 3 1 1 1 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 3 1 3 2 1 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 1 1 2 3 2 1 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 1 2 3 2 2 3 1 3 2 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 3 1 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 2 1 2 2 3 2 1 3 1 1 3 2 1 1 1 3 2 2 1 3 1 1 3 2 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 2 3 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 1 2 1 1 3 1 1 1 2 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 3 1 2 1 2 1 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 3 1 2 2 1 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 2 1 2 3 2 3 2 3 2 2 3 2 2 2 1 3 2 3 2 2 1 2 2 1 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 3 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 2 1 3 2 1 2 2 1 3 2 1 1 3 2 1 2 3 2 3 1 2 2 3 2 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 1 1 2 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 3 1 2 2 3 1 1 2 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 2 1 1 3 2 2 1 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 2 2 2 1 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 3 1 2 1 3 1 2 2 2 1 3 1 1 2 3 1 1 2 2 1 1 3 2 3 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 1 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 2 3 2 2 2 1 2 3 2 1 3 2 3 2 1 3 1 2 2 3 1 1 2 2 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 2 1 3 2 1 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 2 1 3 2 2 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 3 2 1 1 2 3 1 2 1 1 2 3 1 1 3 2 3 2 1 2 1 2 1 3 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 2 1 1 2 3 1 1 3 1 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 2 1 3 2 2 1 3 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 3 1 1 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 1 3 2 2 1 2 1 1 3 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 2 3 2 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 3 1 3 2 2 1 1 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 1 3 2 1 2 1 2 1 3 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 2 1 2 3 2 1 1 1 3 1 1 1 3 2 3 1 1 1 3 1 1 3 1 1 2 3 1 1 2 3 2 1 3 1 1 1 2 3 1 1 2 3 2 2 3 1 1 1 1 1 2 2 3 1 1 2 1 3 2 3 2 3 2 3 1 3 2 2 2 1 1 2 1 3 1 2 1 2 2 3 2 2 2 3 1 2 2 1 1 2 3 1 1 3 1 3 1 1 1 3 2 2 3 2 1 1 1 3 2 2 3 1 1 3 1 2 1 1 1 3 3 2 2 1 1 3 1 3 1 2 2 1 2 3 1 3 1 2 3 2 1 2 2 1 1 3 1 1 3 1 2 1 2 1 1 3 1 1 3 1 2 2 3 1 1 2 2 3 3 2 1 3 1 1 1 2 2 2 3 1 1 2 2 3 1 2 3 2 3 1 1 1 1 1 3 1 3 2 1 3 1 2 2 3 1 2 1 1 3 2 1 2 1 2 3 1 2 3 1 2 1 2 1 3 2 1 3 2 3 1 1 3 1 1 1 2 1 1 3 2 1 3 1 2 1 1 2 3 1 2 3 1 3 1 1 1 2 3 1 1 3 1 2 1 1 2 3 2 3 1 1 1 3 2 1 2 2 2 3 2 3 1 2 1 2 1 3 2 1 1 2 1 1 3 1 3 1 1 2 2 3 1 2 1 2 3 1 1 3 1 2 3 2 1 1 3 2 3 2 1 2 2 2 1 3 2 1 3 1 1 2 3 1 1 3 2 2 1 2 3 2 2 1 3 1 2 2 2 3 2 2 3 1 3 1 2 2 3 1 2 1 3 2 2 2 3 2 1 2 3 1 1 3 1 3 1 2 1 3 2 1 2 2 2 3 1 3 1 1 1 2 3 2 2 1 2 3 2 1 2 2 2 1 3 2 1 3 2 2 1 2 3 2 3 1 3 1 1 2 3 2 3 2 2 2 3 1 2 2 2 1 1 3 2 1 2 3 2 2 2 3 2 2 2 1 2 1 3 1 1 2 3 2 1 2 3 3 1 3 2 1 2 1 2 1 3 1 1 3 1 1 1 3 1 1 1 2 2 2 3 1 2 3 1 3 2 3 1 1 3 2 1 1 1 2 3 2 1 3 2 2 1 2 2 2 2 1 1 3 1 1 3 2 3 1 3 2 2 1 2 2 3 2 3 1 2 1 2 1 2 3 1 1 1 2 3 1 3 1 1 2 1 2 2 3 2 2 3 2 2 2 3 3 1 2 2 1 1 2 3 1 2 2 1 2 3 2 3 1 1 2 2 3 1 2 3 3 1 1 1 2 3 2 2 1 1 1 3 1 2 1 2 3 1 1 1 3 2 1 3 2 1 2 2 3 2 2 3 1 2 2 2 3 1 2 1 2 2 1 3 2 3 2 3 2 2 2 1 2 3 2 2 2 3 2 3 2 1 2 3 2 1 1 3 2 1 3 2 1 1 2 2 3 1 1 1 3 1 1 2 2 3 2 3 2 3 1 1 2 2 3 1 2 3 1 3 2 2 2 3 1 1 2 2 2 3 2 2 2 3 1 3 2 1 1 2 3 1 2 3 2 1 2 1 1 2 3 1 2 3 2 3 2 3 2 1 1 1 2 2 1 2 3 2 3 1 3 1 3 1 1 3 1 1 2 2 2 3 2 2 2 1 2 2 3 2 3 1 2 1 1 1 3 2 1 2 2 3 2 2 3 1 2 1 3 1 1 1 3 1 1 3 2 1 3 1 1 2 1 3 1 1 1 3 2 2 1 1 2 1 3 1 2 2 3 2 3 2 1 3 2 2 1 1 3 1 3 2 2 3 2 2 2 1 1 2 2 1 3 2 1 3 2 1 1 3 2 2 3 2 2 1 3 1 1 2 1 3 2 2 1 1 2 2 2 3 1 1 3 2 1 2 1 1 2 3 1 1 2 3 2 3 2 3 2 1 3 1 1 1 2 2 3 2 1 3 2 1 2 2 2 3 1 3 1 3 1 1 2 3 2 1 2 1 2 3 2 2 1 1 2 3 1 3 1 2 3 2 2 3 2 1 2 1 2 2 2 3 1 2 1 1 3 1 3 1 1 2 3 1 1 3 1 1 3 2 2 2 3 1 1 2 1 3 2 3 2 1 1 2 3 1 1 2 1 2 3 1 2 3 3 2 1 3 2 2 2 3 2 3 1 1 2 1 3 1 1 2 2 1 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 2 1 1 2 2 2 3 2 3 2 3 1 1 3 1 1 3 1 2 2 3 2 2 3 1 3 2 2 1 1 2 1 3 1 2 1 1 1 3 1 2 2 1 2 3 2 1 3 2 3 1 2 3 2 1 1 1 2 3 2 2 3 1 1 2 2 2 1 3 1 2 3 2 1 3 1 2 1 2 3 1 1 2 3 2 3 1 2 1 3 1 1 3 2 3 2 1 2 2 1 1 3 2 1 1 3 2 2 1 2 1 2 3 1 1 2 2 1 2 3 1 3 1 1 3 1 1 2 1 3 1 3 2 2 2 2 3 2 2 1 2 3 1 1 3 2 3 1 2 2 2 3 2 2 2 3 2 3 2 1 1 1 3 1 2 2 3 2 3 2 2 1 2 1 2 3 1 1 1 2 3 2 2 3 2 3 1 2 1 3 2 1 3 2 2 1 3 1 2 1 2 2 2 3 2 3 1 1 2 2 1 1 3 1 2 1 1 1 3 1 1 3 1 3 1 1 3 2 1 3 1 2 2 3 2 1 3 1 1 2 3 1 1 2 2 2 3 2 1 3 2 1 2 1 1 1 2 1 1 3 1 3 1 3 1 3 1 1 2 3 1 2 2 2 1 3 2 1 1 2 2 1 2 3 2 3 1 1 2 1 3 1 2 2 3 2 2 3 1 1 3 2 2 1 1 3 1 2 2 2 1 2 3 2 3 1 2 1 3 2 1 3 1 3 2 2 2 1 1 1 3 1 2 1 3 2 3 2 2 2 3 2 2 3 2 3 2 2 1 2 1 2 2 3 1 2 2 2 1 2 3 1 1 3 1 3 2 1 2 1 3 2 3 1 1 1 2 2 2 3 1 2 3 1 3 2 1 3 2 2 2 1 1 3 1 3 1 1 2 1 1 1 3 2 2 3 2 2 2 3 1 2 3 2 2 2 3 1 1 2 3 3 1 2 2 3 2 3 1 2 3 1 1 2 1 1 2 3 2 2 1 2 2 3 1 3 1 2 3 1 1 3 1 1 1 2 1 2 3 1 2 1 2 3 1 1 2 1 3 2 2 1 1 1 3 2 2 1 2 2 3 1 1 3 2 3 1 1 3 2 2 3 1 2 2 3 2 1 1 3 1 1 1 2 1 3 1 3 1 2 2 2 3 2 3 2 2 3 1 3 1 2 2 3 1 3 2 2 2 1 1 3 2 1 2 2 1 3 1 2 2 1 3 2 3 1 2 1 1 2 1 3 1 1 2 3 1 2 1 1 1 2 3 2 3 3 1 2 1 1 2 1 3 2 3 1 1 2 2 2 3 1 3 2 2 3 2 1 2 1 3 1 2 1 2 2 2 3 2 1 3 2 1 3 1 1 1 3 2 1 2 3 2 3 2 2 1 2 3 1 1 2 3 2 2 3 1 1 2 2 2 3 1 1 2 3 2 1 2 3 1 1 1 3 1 2 2 2 1 3 2 2 3 2 3 1 3 1 2 1 2 1 1 1 2 1 3 1 3 1 1 3 2 2 1 2 3 1 2 3 2 3 1 2 1 2 2 1 3 2 3 1 3 1 1 1 2 3 2 2 2 1 1 2 3 2 3 1 2 2 3 1 1 3 1 1 2 1 2 3 2 3 1 1 1 2 2 1 3 2 2 2 3 3 2 2 2 3 1 2 1 3 2 2 2 1 1 2 3 1 3 2 1 2 2 3 1 3 2 2 3 2 1 1 3 2 1 1 2 3 1 2 1 1 1 3 2 1 2 3 1 2 1 1 3 1 3 2 1 3 2 1 1 2 2 3 2 2 3 2 2 2 1 3 1 2 2 2 3 1 3 1 3 1 3 2 1 2 3 2 1 2 3 1 2 2 1 2 2 1 2 2 3 1 2 2 3 2 3 1 1 2 2 1 3 1 2 1 3 1 1 3 1 3 1 2 2 1 3 2 1 2 2 2 1 3 2 1 3 2 1 1 2 1 3 1 3 2 1 2 3 2 1 2 2 1 3 1 3 1 2 1 2 2 3 1 1 1 3 2 3 2 1 2 3 2 3 1 1 1 3 2 1 1 2 3 1 2 1 1 1 2 3 1 3 3 2 1 1 2 2 1 3 2 1 1 2 3 1 2 2 2 3 1 1 2 3 1 3 3 2 2 2 1 2 2 3 2 1 1 1 3 1 2 3 2 1 1 3 2 3 1 1 2 1 3 2 1 3 1 1 2 2 3 2 2 3 2 2 1 1 1 3 1 1 2 3 2 1 2 2 3 2 2 1 3 2 2 1 2 3 2 1 3 2 3 2 3 2 1 1 3 1 3 2 3 1 1 1 3 2 2 1 2 1 2 3 1 1 1 3 2 1 2 1 1 2 1 2 1 3 1 1 3 2 2 3 1 2 3 1 3 2 2 2 1 2 3 1 2 2 2 1 3 1 1 3 2 1 1 3 1 1 2 1 1 3 2 3 1 3 2 1 2 3 2 3 2 1 2 1 1 3 1 2 1 2 2 2 3 2 1 1 3 1 1 3 2 1 3 1 1 3 1 3 2 2 3 2 1 2 2 3 2 2 1 2 1 1 3 2 3 2 3 2 2 1 2 2 1 3 2 2 2 1 1 3 2 2 1 3 1 3 2 1 1 1 2 1 2 1 3 2 3 1 2 3 2 3 1 1 1 2 2 3 1 1 2 3 2 2 1 3 1 3 1 1 2 1 3 1 3 2 3 1 2 2 1 2 1 3 2 2 3 1 1 3 2 3 1 3 2 2 1 1 2 3 1 2 2 2 3 2 1 1 1 2 1 1 2 3 2 1 1 1 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1 3 2 1 1 2 3 2 1 2 2 2 3 2 2 1 1 3 1 1 1 2 1 3 2 1 3 1 2 1 1 3 2 3 1 1 2 1 1 3 2 1 1 1 2 2 3 1 1 1 3 2 3 2 1 2 1 3 2 3 1 1 3 1 2 3 2 1 2 3 2 2 2 1 2 2 3 2 2 3 2 3 2 1 1 2 2 2 1 3 1 1 2 1 2 1 3 2 3 1 1 3 1 3 1 2 1 3 3 2 2 1 2 3 1 1 1 3 1 3 2 1 2 3 2 3 2 2 1 1 1 2 2 1 2 2 1 2 3 2 3 1 1 3 1 1 3 1 1 2 3 1 2 2 1 3 2 1 1 2 1 1 3 2 2 3 1 1 3 1 3 1 1 2 2 3 2 2 3 2 2 3 1 2 3 2 2 2 3 1 2 3 2 1 1 2 2 3 2 2 1 1 1 3 3 2 3 1 1 1 3 1 2 2 2 3 1 3 2 2 2 3 2 1 2 1 1 2 1 3 1 3 1 1 2 1 2 1 3 1 2 2 3 1 3 1 2 2 2 3 2 2 2 2 2 3 1 3 1 2 3 2 3 1 2 3 1 2 1 1 1 3 2 2 1 1 3 2 2 3 2 1 1 1 2 2 3 2 1 3 2 1 1 1 3 1 1 3 2 1 3 2 3 2 2 1 2 3 1 2 3 2 2 3 2 2 2 3 2 1 2 2 1 2 1 2 2 1 2 2 3 2 3 2 1 3 1 2 3 2 1 2 2 1 1 3 1 3 3 2 2 1 3 1 1 1 3 1 2 2 2 1 3 1 1 3 2 2 1 3 2 2 2 2 3 2 3 2 1 2 2 1 1 3 1 3 1 3 2 3 1 1 1 2 1 2 3 2 2 2 1 1 3 1 2 1 3 1 1 1 3 1 3 2 3 1 2 2 2 1 1 1 2 3 1 3 1 1 1 2 1 3 1 2 1 3 2 2 1 2 2 3 2 3 2 3 1 1 2 2 3 1 1 2 1 1 3 1 1 2 2 2 3 2 2 3 2 3 1 1 2 1 1 3 1 2 2 3 1 1 2 2 1 3 2 3 1 3 2 1 1 3 1 1 2 3 2 2 2 3 1 3 1 3 1 2 2 2 1 3 2 1 1 1 3 1 1 3 2 2 2 1 3 1 1 2 1 3 1 1 1 2 3 2 3 2 2 2 3 1 2 1 2 1 1 3 2 1 1 3 2 3 2 2 1 1 3 1 2 2 2 3 1 3 3 2 1 3 2 3 1 1 2 1 1 3 2 2 1 3 2 3 2 2 1 1 2 1 1 1 3 2 3 2 3 2 2 1 1 1 3 2 1 1 1 2 3 2 1 3 1 2 1 3 1 3 1 2 3 2 2 2 1 2 3 2 2 3 2 3 1 1 2 2 1 1 1 3 2 2 3 1 1 2 1 2 2 3 1 2 3 1 2 1 1 3 1 1 3 1 2 3 1 1 2 3 2 3 1 3 1 2 3 2 2 2 1 3 1 1 2 1 1 2 1 1 2 1 1 2 3 1 2 3 2 1 1 3 2 2 2 3 1 3 2 2 2 3 1 1 1 3 1 3 2 3 1 1 2 1 3 1 1 1 2 1 1 3 1 3 1 1 1 1 2 1 1 1 3 2 2 1 2 2 3 1 3 1 3 1 3 2 2 2 1 3 1 3 2 1 3 2 3 2 2 3 2 1 3 2 2 2 1 3 2 1 2 1 2 1 3 2 1 1 3 1 1 2 3 2 1 2 2 1 3 1 2 1 2 2 2 3 2 3 3 1 2 1 1 1 2 3 2 2 2 3 1 2 1 1 1 3 2 1 3 2 2 3 1 2 1 3 2 1 2 3 2 1 2 3 2 3 2 3 1 1 3 1 2 2 2 1 1 2 3 1 1 2 3 2 1 3 1 3 2 3 1 2 2 1 3 2 2 2 1 1 3 2 1 3 2 1 2 2 2 1 3 2 3 1 2 3 2 1 1 3 1 1 2 1 1 3 1 1 2 2 3 2 1 2 2 3 1 1 3 1 1 3 1 1 2 1 2 3 2 2 2 1 2 1 3 1 1 2 2 3 1 3 1 3 1 1 3 2 2 1 1 3 1 1 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 1 3 1 1 2 2 1 2 2 2 1 1 1 3 1 1 1 3 2 1 2 2 3 2 1 1 3 1 3 2 3 1 1 1 2 2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 1 3 2 2 2 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1 2 2 2 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1 1 3 1 3 1 1 2 1 1 2 3 2 1 3 1 3 1 2 1 2 1 1 3 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 1 1 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1 2 2 3 2 3 2 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 3 1 2 2 3 1 2 3 2 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 1 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 2 2 1 1 1 2 3 1 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 1 2 3 1 1 2 1 1 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 2 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 2 2 2 3 1 1 2 3 1 1 1 2 2 3 1 2 3 1 2 1 3 1 2 3 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 3 1 1 1 3 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 2 2 2 1 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 2 1 1 1 3 2 3 1 1 1 3 1 2 2 2 3 1 1 1 2 3 1 2 3 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 2 2 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 2 2 3 2 3 1 1 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 2 2 3 2 3 2 2 3 1 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 2 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 2 2 3 2 1 2 2 2 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 1 3 1 1 1 2 1 2 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 2 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 3 2 2 3 2 1 1 2 2 2 3 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 2 2 3 2 3 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1 3 2 3 1 3 1 2 2 2 1 3 1 1 3 1 2 3 2 2 1 2 2 1 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 3 2 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2

for any pair of sequences of the set:

M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19 where:

M1 is the maximum number of matches for any alignment in which there are no internal indels;

M2 is the maximum length of a block of matches for any alignment;

M3 is the maximum number of matches for any alignment having a maximum score;

M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and

M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein:

the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:

for each of (i) to (iv):

(i) m=6, mm=6, og=0 and eg=6,

(ii) m=6, mm=6, og=0 and eg=1,

(iii) m=6, mm=2, og=5 and eg=1, and

(iv) m=6, mm=6, og=6 and eg=0,

A is the total number of matched pairs of bases in the alignment;

B is the total number of internal mismatched pairs in the alignment;

C is the total number of internal gaps in the alignment; and

D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and

wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv).

c) detecting said second non-target cleavage product.

68. The method of claim 67, wherein said first target nucleic acid is genomic DNA and said second target nucleic acid is synthetic DNA.

69. The method of claim 68, wherein said synthetic DNA has at least one hairpin loop.

70. The method of claim 69, wherein the method includes a plurality of said first target nucleic acid sequences, a plurality of first oligonucleotide molecules, a plurality of said second oligonucleotide molecules, a plurality of said second target nucleic acid sequences and a plurality of third oligonucleotide molecules.

Description

FIELD OF THE INVENTION

[0001]

This invention relates to the use of families of oligonucleotides for use as tags, for example, in the sorting of molecules, identification of target nucleic acid molecules or for analyzing the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule.

BACKGROUND OF THE INVENTION

[0002]

Single-nucleotide polymorphisms (SNPs) are the most common form of genetic polymorphism. This, coupled with their potential as functional variants, has produced a great deal of interest in SNPs both as pharmacogenetic indicators and as markers for mapping genes for complex diseases. A large number of SNPs have already been identified with >21,000 entries on the NCBI's SNP database alone. Many recent studies have focused on identifying polymorphisms that lie in the coding sequence of potential candidate genes for common diseases. The ability to genotype this abundant source of variation rapidly and accurately is becoming an ever more important goal in the genetics community. A variety of technologies have the potential to transfer to high-throughput genotyping laboratories. These include 5′ exonuclease assays, such as TaqMan (Livak et al. 1995), molecular beacons (Tyagi et al. 1996), dye-labeled oligonucleotide ligation (DOL) (Chen et al. 1998), oligonucleotide-ligation assays (OLAs) (To be et al. 1996), minisequencing (Chen and Kwok 1997; Pastinen et al. 1997), microarray technology (Hacia et al. 1998; Wang et al. 1998), mass spectroscopy (Ross et al. 1998) and the scorpions assay (Whitcombe et al. 1999). However, no single chemistry has gained acceptance as the technology of choice. A suitable method for such applications must be accurate and homogenous, develop a robust, easily interpretable signal, and be flexible enough to extend to novel foci with little optimization. These features will lend the technology to automation.
[0003]

Third Wave Technologies, Inc., has developed a new mutation detection method referred to as the Invader Assay. The Invader Assay is based on a novel linear signal amplification technology that requires specific hybridization of two “overlapping” oligonucleotides and subsequent recognition and cleavage of this structure by the Cleavase enzyme. Cleavases are bacterial enzymes that cleave unpaired DNA strands or “flaps” near a nick, for instance when the 5′ end of a sequence is displaced by the 3′ end of an elongating upstream oligonucleotide. Enzymes with this so-called flap endonuclease activity typically excise the redundant 5′ “flap” of the downstream oligonucleotide, leaving a simple nick to be repaired by lipases. The excised “flap” is subsequently detected by one of several methods commonly known in the art. Cleavases have stringent requirements relative to the structure formed by such overlapping DNA sequences, and can be used to specifically detect single base pair mismatches immediately upstream of the cleavage site on the downstream DNA strand. Thermostable cleavages permit reactions to be performed at temperatures sufficiently high to promote turnover and consequent signal amplification without the need for temperature cycling.
[0004]

While the Invader Assay offers exquisite specificity, its use in the detection of multiple distinct target nucleic acids in a single experiment i.e., multiplexing, is limited. This is because if the Invader Assay is to be used in a high-throughput gene microarray format, the most efficient method of detecting the excised “flap” sequence is to capture the sequence by hybridization to its complementary nucleic acid sequence attached to a solid phase support.
[0005]

Working in a highly parallel hybridization environment requiring specific hybridization imposes very rigorous selection criteria for the design of families of oligonucleotides that are to be used. The success of these approaches is dependent on the specific hybridization of a probe and its complement. Problems arise as the family of nucleic acid molecules cross-hybridise or hybridise incorrectly to the target sequences. While it is common to obtain incorrect hybridization resulting in false positives or an inability to form hybrids resulting in false negatives, the frequency of such results must be minimized. In order to achieve this goal certain thermodynamic properties of forming nucleic acid hybrids must be considered. The temperature at which oligonucleotides form duplexes with their complementary sequences known as the T. (the temperature at which 50% of the nucleic acid duplex is dissociated) varies according to a number of sequence dependent properties including the hydrogen bonding energies of the canonical pairs A-T and G-C (reflected in GC or base composition), stacking free energy and, to a lesser extent, nearest neighbour interactions. These energies vary widely among oligonucleotides that are typically used in hybridization assays. For example, hybridization of two probe sequences composed of 24 nucleotides, one with a 40% GC content and the other with a 60% GC content, with its complementary target under standard conditions theoretically may have a 10° C. difference in melting temperature (Mueller et al., Current Protocols in Mol. Biol.; 15, 5:1993). Problems in hybridization occur when the hybrids are allowed to form under hybridization conditions that include a single hybridization temperature that is not optimal for correct hybridization of all oligonucleotide sequences of a set. Mismatch hybridization of non-complementary probes can occur forming duplexes with measurable mismatch stability (Santalucia et al., Biochemistry; 38: 3468-77, 1999). Mismatching of duplexes in a particular set of oligonucleotides can occur under hybridization conditions where the mismatch results in a decrease in duplex stability that results in a higher T_mthan the least stable correct duplex of that particular set. For example, if hybridization is carried out under conditions that favor the AT-rich perfect match duplex sequence, the possibility exists for hybridizing a GC-rich duplex sequence that contains a mismatched base having a melting temperature that is still above the correctly formed AT-rich duplex. Therefore, design of families of oligonucleotide sequences that can be used in multiplexed hybridization reactions must include consideration for the thermodynamic properties of oligonucleotides and duplex formation that will reduce or eliminate cross hybridization behavior within the designed oligonucleotide set.
[0006]

The development of such families of tags has been attempted over the years with varying degrees of success. There are a number of different approaches for selecting sequences for use in multiplexed hybridization assays. The selection of sequences that can be used as zipcodes or tags in an addressable array has been described in the patent literature in an approach taken by Brenner and co-workers. U.S. Pat. No. 5,654,413 describes a population of oligonucleotide tags (and corresponding tag complements) in which each oligonucleotide tag includes a plurality of subunits, each subunit consisting of an oligonucleotide having a length of from three to six nucleotides and each subunit being selected from a minimally cross hybridizing set, wherein a subunit of the set would have at least two mismatches with any other sequence of the set. Table II of the Brenner patent specification describes exemplary groups of 4mer subunits that are minimally cross hybridizing according to the aforementioned criteria. In the approach taken by Brenner, constructing non cross-hybridizing oligonucleotides, relies on the use of subunits that form a duplex having at least two mismatches with the complement of any other subunit of the same set. The ordering of subunits in the construction of oligonucleotide tags is not specifically defined.
[0007]

Parameters used in the design of tags based on subunits are discussed in Barany et al. (WO 9731256). For example, in the design of polynucleotide sequences that are for example 24 nucleotides in length (24mer) derived from a set of four possible tetramers in which each 24mer “address” differs from its nearest 24mer neighbour by 3 tetramers. They discuss further that, if each tetramer differs from each other by at least two nucleotides, then each 24mer will differ from the next by at least six nucleotides. This is determined without consideration for insertions or deletions when forming the alignment between any two sequences of the set. In this way a unique “zip code” sequence is generated. The zip code is ligated to a label in a target dependent manner, resulting in a unique “zip code” which is then allowed to hybridise to its address on the chip. To minimise cross-hybridisation of a “zip code” to other “addresses”, the hybridization reaction is carried out at temperatures of 75-80° C. Due to the high temperature conditions for hybridization, 24mers that have partial homology hybridise to a lesser extent than sequences with perfect complementarity and represent ‘dead zones’. This approach of implementing stringent hybridization conditions for example, involving high temperature hybridization, is also practiced by Brenner et. al.
[0008]

The current state of technology for designing non-cross hybridizing tags based on subunits does not provide sufficient guidance to construct a family of relatively large numbers of sequences with practical value in assays that require stringent non-cross hybridizing behavior.
[0009]

Thus, while it is desirable to have, at once in a gene microarray format, a large number of “flap” molecules incorporated into the Invader Assay, the “flap” molecules should each be highly selective for its own complement sequence. While such an array provides the advantage that the family of molecules making up the grid is entirely of design, and does not rely on sequences as they occur in nature, the provision of a family of molecules, which is, sufficiently large and where each individual member is sufficiently selective for its complement over all the other zipcode molecules (i.e., where there is sufficiently low cross-hybridization, or cross-talk) continues to elude researchers.

SUMMARY OF THE INVENTION

[0010]

The present invention relates to the use of one set of 210 and a second set of 1168 minimally cross-hybridizing oligonucleotide sequences for use in the Invader Assay. The incorporation of these sequences into one of the two probes, and subsequent structure dependent cleavage of the minimally cross-hybridizing sequences upon hybridization to the target nucleic acid molecule enables the Invader Assay to be used in the analysis of multiple gene n a gene microarray.
[0011]

Using the method of Benight et al. a family of 100 sequences was obtained using a computer algorithm to have optimal hybridization properties for use in nucleic acid detection assays. The sequence set of 100 oligonucleotides was characterized in hybridization assays, demonstrating the ability of family members to correctly hybridize to their complementary sequences with an absence of cross hybridization. These are the sequences having SEQ ID NOs:1 to 100 of Table I. This set of sequences has been expanded to include an additional 110 sequences that can be grouped with the original 100 sequences as having non-cross hybridizing properties, based on the characteristics of the original set of 100 sequences. These additional sequences are identified as SEQ ID NOs:101 to 210 of the sequences in Table I. How these sequences were obtained is described below.
[0012]

Variant families of sequences (seen as tags or tag complements) of a family of sequences taken from Table I are also part of the invention. For the purposes of discussion, families of tag complements will be described.
[0013]

A family of complements is obtained from a set of oligonucleotides based on a family of oligonucleotides such as those of Table I. For illustrative purposes, providing a family of complements based on the oligonucleotides of Table I will be described.

[0014]

Firstly, the groups of sequences based on the oligonucleotides of Table I can be represented as follows:

TABLE IA


Numeric sequences corresponding to word
patterns of a set of oligonucleotides
Sequence
Identifier	Numeric Pattern

1	1	4	6	6	1	3
2	2	4	5	5	2	3
3	1	8	1	2	3	4
4	1	7	1	9	8	4
5	1	1	9	2	6	9
6	1	2	4	3	9	6
7	9	8	9	8	10	9
8	9	1	2	3	8	10
9	8	8	7	4	3	1
10	1	1	1	1	1	2
11	2	1	3	3	2	2
12	3	1	2	2	3	2
13	4	1	4	4	4	2
14	1	2	3	3	1	1
15	1	3	2	2	1	4
16	3	3	3	3	3	4
17	4	3	1	1	4	4
18	3	4	1	1	3	3
19	3	6	6	6	3	5
20	6	6	1	1	6	5
21	7	6	7	7	7	5
22	8	7	5	5	8	8
23	2	1	7	7	1	1
24	2	3	2	3	1	3
25	2	6	5	6	1	6
26	4	8	1	1	3	8
27	5	3	1	1	6	3
28	5	6	8	8	6	6
29	8	3	6	5	7	3
30	1	2	3	1	4	6
31	1	5	7	5	4	3
32	2	1	6	7	3	6
33	2	6	1	3	3	1
34	2	7	6	8	3	1
35	3	4	3	1	2	5
36	3	5	6	1	2	7
37	3	6	1	7	2	7
38	4	6	3	5	1	7
39	5	4	6	3	8	6
40	6	8	2	3	7	1
41	7	1	7	8	6	3
42	7	3	4	1	6	8
43	4	7	7	1	2	4
44	3	6	5	2	6	3
45	1	4	1	4	6	1
46	3	3	1	4	8	1
47	8	3	3	5	3	8
48	1	3	6	6	3	7
49	7	3	8	6	4	7
50	3	1	3	7	8	6
51	10	9	5	5	10	10
52	7	10	10	10	7	9
53	9	9	7	7	10	9
54	9	3	10	3	10	3
55	9	6	3	4	10	6
56	10	4	10	3	9	4
57	3	9	3	10	4	9
58	9	10	5	9	4	8
59	3	9	4	9	10	7
60	3	5	9	4	10	8
61	4	10	5	4	9	3
62	5	3	3	9	8	10
63	6	8	6	9	7	10
64	4	6	10	9	6	4
65	4	9	8	10	8	3
66	7	7	9	10	5	3
67	8	8	9	3	9	10
68	8	10	2	9	5	9
69	9	6	2	2	7	10
70	9	7	5	3	10	6
71	10	3	6	8	9	2
72	10	9	3	2	7	3
73	8	9	10	3	6	2
74	3	2	5	10	8	9
75	8	2	3	10	2	9
76	6	3	9	8	2	10
77	3	7	3	9	9	10
78	9	10	1	1	9	4
79	10	1	9	1	4	1
80	7	1	10	9	8	1
81	9	1	10	1	10	6
82	9	6	9	1	3	10
83	3	10	8	8	9	1
84	3	8	1	9	10	3
85	9	10	1	3	6	9
86	1	9	1	10	3	1
87	1	4	9	6	8	10
88	3	3	9	6	1	10
89	5	3	1	6	9	10
90	6	1	8	10	9	6
91	5	9	9	4	10	3
92	2	10	9	1	9	5
93	10	10	7	2	1	9
94	10	9	9	1	8	2
95	1	8	6	8	9	10
96	1	9	1	3	8	10
97	9	6	9	10	1	2
98	1	10	8	9	9	2
99	1	9	6	7	2	9
100	4	3	9	3	5	1
101	5	11	10	14	12	1
102	7	12	4	13	3	2
103	5	5	4	4	12	9
104	2	13	13	11	13	13
105	10	2	5	4	12	7
106	11	7	4	11	6	4
107	12	12	1	9	11	11
108	12	9	4	14	12	6
109	12	7	13	2	9	11
110	9	11	3	4	1	3
111	10	5	12	11	4	4
112	4	13	7	12	1	5
113	9	13	10	11	11	6
114	10	14	14	10	1	3
115	2	14	1	10	4	5
116	10	12	12	7	11	10
117	9	11	2	12	8	11
118	2	8	5	2	12	14
119	1	8	13	3	7	8
120	9	4	7	5	4	2
121	13	2	12	7	1	12
122	11	10	9	7	5	11
123	8	12	2	2	12	7
124	5	2	14	3	4	13
125	1	8	8	1	5	9
126	14	5	11	10	13	3
127	14	1	4	13	2	4
128	4	4	5	11	3	10
129	10	9	2	3	3	11
130	11	4	8	14	3	4
131	5	1	14	8	11	2
132	14	3	11	6	12	5
133	13	4	4	1	10	1
134	6	10	11	6	5	1
135	5	8	12	5	1	7
136	4	5	9	6	9	2
137	13	2	4	4	2	3
138	11	2	2	5	9	3
139	8	1	10	12	2	8
140	12	7	9	11	4	1
141	12	1	4	14	3	13
142	11	2	7	10	4	1
143	3	4	12	11	11	11
144	3	3	4	2	12	11
145	1	5	9	4	2	1
146	6	1	12	2	10	5
147	10	5	1	12	2	14
148	2	11	7	9	4	11
149	7	4	4	5	14	12
150	12	5	2	1	10	12
151	5	9	2	11	6	1
152	12	14	3	6	1	14
153	5	9	11	10	1	4
154	2	5	12	14	10	10
155	4	5	8	4	5	6
156	10	12	4	6	12	5
157	4	2	1	13	6	8
158	9	10	10	14	5	3
159	6	14	10	11	3	3
160	2	9	10	12	5	7
161	13	3	7	10	5	12
162	6	4	1	2	5	13
163	6	1	13	4	14	13
164	2	12	1	14	1	9
165	4	11	13	2	6	10
166	1	10	7	4	5	8
167	7	2	2	10	13	4
168	8	2	11	4	6	14
169	4	8	2	6	2	3
170	7	1	12	11	2	9
171	5	6	10	4	13	4
172	5	10	4	11	9	3
173	3	11	9	3	2	3
174	8	15	6	20	17	19
175	21	10	15	3	7	11
176	11	7	17	20	14	9
177	16	6	17	13	21	21
178	10	15	22	6	17	21
179	15	7	17	10	22	22
180	3	20	8	15	20	16
181	17	21	10	16	6	22
182	6	21	14	14	14	16
183	7	17	3	20	10	7
184	16	19	14	17	7	21
185	20	16	7	15	22	10
186	20	10	18	11	22	18
187	18	7	19	15	7	22
188	21	18	7	21	16	3
189	14	13	7	22	17	13
190	19	7	8	12	10	17
191	15	3	21	14	9	7
192	19	6	15	7	14	14
193	4	17	10	15	20	19
194	21	6	18	4	20	16
195	2	19	8	17	6	13
196	12	12	6	17	4	20
197	16	21	12	10	19	16
198	14	14	15	2	7	21
199	8	16	21	6	22	16
200	14	17	22	14	17	20
201	10	21	7	15	21	18
202	16	13	20	18	21	12
203	15	7	4	22	14	13
204	7	19	14	8	15	4
205	4	5	3	20	7	16
206	22	18	6	18	13	20
207	19	6	16	3	13	3
208	18	6	22	7	20	18
209	10	17	11	21	8	13
210	7	10	17	19	10	14

Here, each of the numerals 1 to 22 (numeric identifiers) represents a 4mer and the pattern of numerals 1 to 10 of the sequences in the above list corresponds to the pattern of tetrameric oligonucleotide segments present in the oligonucleotides of Table I, which oligonucleotides have been found to be non-cross-hybridizing, as described further in the detailed examples. Each 4mer is selected from the group of 4mers consisting of WWWW, WWWX, WWWY, WWXW, WWXX, WWXY, WWYW, WWYX, WWYY, WXWW, WXWX, WXWY, WXXW, WXXX, WXXY, WXYW, WXYX, WXYY, WYWW, WYWX, WYWY, WYXW, WYXX, WYXY, WYYW, WYYX, WYYY, XWWW, XWWX, XWWY, XWXW, XWXX, XWXY, XWYW, XWYX, XWYY, XXWW, XXWX, XXWY, XXXW, XXXX, XXXY, XXYW, XXYX, XXYY, XYWW, XYWX, XYWY, XYXW, XYXX, XYXY, XYYW, XYYX, XYYY, YWWW, YWWX, YWWY, YWXW, YWXX, YWXY, YWYW, YWYX, YWYY, YXWW, YXWX, YXWY, YXXW, YXXX, YXXY, YXYW, YXYX, YXYY, YYWW, YYWX, YYWY, YYXW, YYXX, YYXY, YYYW, YYYX, and YYYY. Here W, X and Y represent nucleotide bases, A, G, C, etc., the assignment of bases being made according to rules described below.

[0015]

Given this numeric pattern, a 4mer is assigned to a numeral. For example, 1=WXYY, 2=YWXY, etc. Once a given 4mer has been assigned to a given numeral, it is not assigned for use in the position of a different numeral. It is possible, however, to assign a different 4mer to the same numeral. That is, for example, the numeral 1 in one position could be assigned WXYY and another numeral 1, in a different position, could be assigned XXXW, but none of the other numerals 2 to 10 can then be assigned WXYY or XXXW. A different way of saying this is that each of 1 to 10 is assigned a 4mer from the list of eighty-one 4mers indicated so as to be different from all of the others of 1 to 10.
[0016]

In the case of the specific oligonucleotides given in Table I, 1=WXYY, 2=YWXY, 3=XXXW, 4=YWYX, 5=WYXY, 6=YYWX, 7=YWXX, 8=WYXX, 9=XYYW, 10=XYWX, 11=YYXW, 12=WYYX, 13=XYXW, 14=WYYY, 15=WXYW, 16=WYXW, 17=WXXW, 18=WYYW, 19=XYYX, 20=YXYX, 21=YXXY and 22=XYXY.
[0017]
Once the 4mers are assigned to positions according to the above pattern, a particular set of oligonucleotides can be created by appropriate assignment of bases, A, T/U, G, C to W, X, Y. These assignments are made according to one of the following two sets of rules:
- (i) Each of W, X and Y is a base in which:
  - (a) W=one of A, T/U, G, and C,
    - X=one of A, T/U, G, and C,
    - Y=one of A, T/U, G, and C,
    - and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,
  - (b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y.
    or
- (ii) Each of W, X and Y is a base in which:
  - (a) W=G or C,
    - X=A or T/U,
    - Y=A or T/U,
    - and X≠Y, and
  - (b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in each sequence as that of every o sequence of the set;
[0030]

In the case of the specific oligonucleotides given in Table I, W=G, X=A and Y=T.
[0031]

In any case, given a set of oligonucleotides generated according to one of these sets of rules, it is possible to modify the members of a given set in relatively minor ways and thereby obtain a different set of sequences while more or less maintaining the cross-hybridization properties of the set subject to such modification. In particular, it is possible to insert up to 3 of A, T/U, G and C at any location of any sequence of the set of sequences. Alternatively, or additionally, up to 3 bases can be deleted from any sequence of the set of sequences.
[0032]

A person skilled in the art would understand that given a set of oligonucleotides having a set of properties making it suitable for use as a family of tags (or tag complements) one can obtain another family with the same property by reversing the order of all of the members of the set. In other words, all the members can be taken to be read 51 to 31 or to be read 3′ to 5′.
[0033]

A family of complements of the present invention is based on a given set of oligonucleotides defined as described above. Each complement of the family is based on a different oligonucleotide of the set and each complement contains at least 10 consecutive (i.e., contiguous) bases of the oligonucleotide on which it is based. For a given family of complements where one is seeking to reduce or minimize inter-sequence similarity that would result in cross-hybridization, each and every pair of complements meets particular homology requirements. Particularly, subject to limited exceptions, described below, any two complements within a set of complements are generally required to have a defined amount of dissimilarity.
[0034]

In order to notionally understand these requirements for dissimilarity as they exist for a given pair of complements of a family, a phantom sequence is generated from the pair of complements. A “phantom” sequence is a single sequence that is generated from a pair of complements by selection, from each complement of the pair, of a string of bases wherein the bases of the string occur in the same order in both complements. An object of creating such a phantom sequence is to create a convenient and objective means of comparing the sequence identity of the two parent sequences from which the phantom sequence is created.

[0035]

A phantom sequence may thus be generated from exemplary Sequence 1 and Sequence 2 as follows:


	Sequence 1:	ATGTTTAGTGAAAAGTTAGTATTG
		* •

	Sequence 2:	ATGTTAGTGAATAGTATAGTATTG
		• ♦

	Phantom Sequence:	ATGTTAGTGAAAGTTAGTATTG

[0036]
The phantom sequence generated from these two sequences is thus 22 bases in length. That is, one can see that there are 22 identical bases with identical sequence (the same order) in Sequence Nos. 1 and 2. There is a total of three insertions/deletions and mismatches present in the phantom sequence when compared with the sequences from which it was generated:
- ATGT-TAGTGAA-AGT-TAGTATTG
  The dashed lines in this latter representation of the phantom sequence indicate the locations of the insertions/deletions and mismatches in the phantom sequence relative to the parent sequences from which it was derived. Thus, the “T” marked with an asterisk in Sequence 1, the “A” marked with a diamond in Sequence 2 and the “A-T” mismatch of Sequences 1 and 2 marked with two dots were deleted in generating the phantom sequence.
[0038]

A person skilled in the art will appreciate that the term “insertion/deletion” is intended to cover the situations indicated by the asterisk and diamond. Whether the change is considered, strictly speaking, an insertion or deletion is merely one of vantage point. That is, one can see that the fourth base of Sequence 1 can be deleted therefrom to obtain the phantom sequence, or a “T” can be inserted after the third base of the phantom sequence to obtain Sequence 1.
[0039]

One can thus see that if it were possible to create a phantom sequence by elimination of a single insertion/deletion from one of the parent sequences, that the two parent sequences would have identical homology over the length of the phantom sequence except for the presence of a single base in one of the two sequences being compared. Likewise, one can see that if it were possible to create a phantom sequence through deletion of a mismatched pair of bases, one base in each parent, that the two parent sequences would have identical homology over the length of the phantom sequence except for the presence of a single base in each of the sequences being compared. For this reason, the effect of an insertion/deletion is considered equivalent to the effect of a mismatched pair of bases when comparing the homology of two sequences.
[0040]

Once a phantom sequence is generated, the compatibility of the pair of complements from which it was generated within a family of complements can be systematically evaluated:
[0041]
According to one embodiment of the invention, a pair of complements is compatible for inclusion within a family of complements if any phantom sequence generated from the pair of complements has the following properties:
- - Any consecutive sequence of bases in the phantom sequence which is identical to a consecutive sequence of bases in each of the first and second complements from which it is generated is no more ((¾×L)−1) bases in length;
  - The phantom sequence, if greater than or equal to (⅚×L) in length, contains at least 3 insertions/deletions or mismatches when compared to t first and second complements from which it is generated; and
  - The phantom sequence is not greater than or equal to ( 11/12×L) in length.
[0045]

Here, L₁is the length of the first complement, L₂is the length of the second complement, and L=L₁, or if L₁≠L₂, L is the greater of L₁and L₂.
[0046]

In particular preferred embodiments of the invention, all pairs of complements of a given set have the properties set out above. Under particular circumstances, it may be advantageous to have a limited number of complements that do not meet all of these requirements when compared to every other complement in a family.
[0047]

In one case, for any first complement there are at most two second complements in the family which do not meet all of the three listed requirements. For two such complements, there would thus be a greater chance of cross-hybridization between their tag counterparts and the first complement. In another case, for any first complement there is at most one second complement which does not meet all of three listed requirements.
[0048]

It is also possible, given this invention, to design a family of complements where a specific number or specific portion of the complements do not meet the three listed requirements. For example, a set could be designed where only one pair of complements within the set do not meet the requirements when compared to each other. There could be two pairs, three pairs, and any number of pairs up to and including all possible pairs. Alternatively, it may be advantageous to have a given proportion of pairs of complements that do not meet the requirements, say 10% of pairs, when compared with other sequences that do not meet one or more of the three requirements listed. This number could instead by 5%, 15%, 20%, 25%, 30%, 35%, or 40%.
[0049]

The foregoing comparisons would generally be largely carried out using appropriate computer software. Although notionally described in terms of a phantom sequence for the sake of clarity and understanding, it will be understood that a competent computer programmer can carry out pairwise comparisons of complements in any number of ways using logical steps that obtain equivalent results.
[0050]

The symbols A, G, T/U; C take on their usual meaning in the art here. In the case of T and U, a person skilled in the art would understand that these are equivalent to each other with respect to the inter-strand hydrogen-bond (Watson-Crick) binding properties at work in the context of this invention. The two bases are thus interchangeable and hence the designation of T/U.
[0051]

Analogues of the naturally occurring bases can be inserted in their respective places where desired. Analogues can be defined as any non-natural base, such as peptide nucleic acids and the like.
[0052]

Other aspects of the invention are described below, particularly numbered paragraphs at the end of this specification.
[0053]

In another broad embodiment A family of 1168 sequences was obtained using a computer algorithm to have desirable hybridization properties for use in nucleic acid detection assays. The sequence set of 1168 oligonucleotides was partially characterized in hybridization assays, demonstrating the ability of family members to correctly hybridize to their complementary sequences with minimal cross hybridization. These are the sequences having SEQ ID NOs:1 to 1168 of Table II.
[0054]

Variant families of sequences (seen as tags or tag complements) of a family of sequences taken from Table II are also part of the invention. For the purposes of discussion, a family or set of oligonucleotides will-often be described as a family of tag complements, but it will be understood that such a set could just easily be a family of tags.
[0055]

A family of complements is obtained from a set of oligonucleotides based on a family of oligonucleotides such as those of Table II. To simplify discussion, providing a family of complements based on the oligonucleotides of Table II will be described.

[0056]

Firstly, the groups of sequences based on the oligonucleotides of Table II can be represented as shown in Table IIA.

TABLE IIA


Numeric sequences corresponding to nucleotide patterns of a set of oligonucleotides
	Sequence
Numeric Pattern	Identifier

1	1	1	2	2	3	2	3	1	1	1	3	1	2	2	3	2	2	2	3	2	3	2	1	1
3	2	2	1	3	1	3	2	2	1	1	2	2	3	2	1	2	2	2	3	1	2	3	1	2
1	2	3	2	2	1	1	1	3	2	1	1	3	2	3	2	2	3	1	1	1	2	3	2	3
2	3	1	2	3	2	2	1	3	1	1	3	2	1	2	1	2	2	3	2	3	1	1	2	4
2	2	2	3	2	3	2	1	3	1	1	2	1	2	3	2	3	2	2	3	2	2	1	1	5
1	2	1	1	3	2	3	2	1	1	3	2	3	1	1	1	2	1	1	3	1	1	3	1	6
1	1	3	1	3	2	1	2	2	2	3	2	2	3	2	3	1	3	2	2	1	1	1	2	7
3	2	3	2	2	2	1	2	3	2	2	1	2	1	2	3	2	3	1	1	3	2	2	2	8
1	1	1	3	1	3	1	1	2	1	3	1	1	2	1	2	3	2	3	2	1	1	3	2	9
2	1	2	3	1	1	1	3	1	3	2	3	1	3	1	2	1	1	2	3	2	2	2	1	10
1	2	3	1	3	1	1	1	2	1	2	3	2	2	1	3	1	1	2	3	2	3	1	2	11
2	2	1	3	2	2	3	2	2	3	1	2	3	2	2	2	1	3	2	1	3	2	2	2	12
3	2	1	1	1	3	1	3	2	1	2	1	1	3	2	2	2	3	1	2	3	1	2	1	13
1	1	1	3	2	1	1	3	1	1	2	3	1	2	3	2	1	1	2	1	1	3	2	3	14
3	2	1	3	1	1	1	2	1	3	2	2	2	1	2	2	3	1	2	3	1	2	2	3	15
2	3	2	1	1	3	2	3	1	1	1	2	1	3	2	3	1	3	2	2	1	2	2	2	16
1	1	1	2	1	3	1	2	3	1	2	1	2	1	1	3	2	3	1	3	1	1	2	3	17
1	2	1	1	3	2	2	1	2	1	1	3	2	3	2	2	1	2	3	2	3	1	3	2	18
2	1	2	1	3	1	2	1	1	1	3	1	3	1	2	3	1	2	2	2	3	2	2	3	19
1	3	1	3	2	2	3	1	3	1	1	2	3	2	1	2	1	3	2	1	2	2	1	2	20
1	1	3	2	1	3	2	2	2	3	2	1	1	3	1	1	2	3	1	2	2	3	2	1	21
2	2	1	2	3	1	1	1	2	2	3	1	3	2	3	1	1	3	1	2	2	3	1	2	22
3	2	1	2	1	2	3	2	1	1	1	2	2	3	2	2	1	2	3	2	2	3	1	3	23
3	1	1	2	2	3	2	1	2	1	1	1	3	2	1	2	2	1	3	1	2	3	2	3	24
2	1	3	1	2	3	1	3	1	2	2	1	1	3	2	3	2	2	1	2	2	2	3	1	25
3	2	2	1	1	3	2	2	2	3	2	2	2	1	2	3	2	1	2	1	3	1	1	3	26
3	1	3	2	1	2	2	1	3	2	1	1	1	3	2	3	1	2	1	2	3	1	2	1	27
3	2	3	1	1	2	3	1	2	2	2	1	3	2	1	1	1	2	3	1	2	2	3	1	28
3	1	2	2	3	1	1	3	2	2	1	2	1	3	1	1	1	2	3	1	2	2	1	3	29
1	3	2	3	1	2	1	1	1	2	3	2	2	1	3	2	2	3	1	1	2	2	3	2	30
2	1	2	1	2	1	3	2	1	1	1	2	3	2	2	2	3	2	3	2	3	2	2	3	31
2	2	1	1	3	2	3	2	2	1	3	2	2	1	2	2	2	3	2	2	3	2	1	3	32
3	2	1	3	2	1	1	2	1	2	3	1	1	3	2	3	1	3	1	1	2	1	2	1	33
2	1	3	2	3	2	1	2	1	3	1	1	2	3	2	1	3	1	2	2	2	1	3	2	34
2	2	3	2	1	3	1	2	2	1	3	1	2	3	2	3	2	2	2	3	2	1	1	1	35
2	1	3	2	1	2	1	3	1	3	2	1	3	1	3	1	2	3	1	2	1	2	2	2	36
1	2	2	3	2	3	1	1	1	3	1	1	1	3	1	3	1	1	3	1	1	1	2	2	37
2	3	2	3	1	3	1	1	2	2	1	1	3	1	2	2	1	1	3	1	1	2	3	2	38
1	2	1	2	2	1	3	2	2	1	1	3	1	1	1	3	1	1	3	1	3	2	2	3	39
2	2	3	2	1	3	2	2	3	1	3	1	1	1	2	1	2	3	2	1	3	2	2	2	40
2	1	3	1	3	2	2	3	2	2	1	1	1	3	1	3	2	3	2	1	1	1	2	1	41
3	2	2	1	2	3	1	2	3	2	3	2	1	2	1	1	3	2	1	1	2	1	2	3	42
2	2	2	3	2	2	1	3	1	1	2	3	1	3	1	1	3	1	2	2	2	1	2	3	43
1	3	2	1	2	1	3	2	2	2	1	1	1	3	1	1	3	2	1	3	2	1	3	1	44
3	2	3	1	3	1	2	1	2	1	3	1	2	2	2	1	3	1	1	1	3	2	1	1	45
2	2	3	2	2	2	1	2	1	3	2	3	1	1	3	2	3	1	1	2	1	3	2	1	46
1	1	3	2	1	1	3	2	1	3	2	1	1	2	1	3	2	3	2	3	2	2	1	1	47
1	2	2	2	3	2	3	1	3	2	2	1	2	3	1	1	1	3	1	2	1	1	3	1	48
3	1	1	1	3	2	1	3	1	3	1	1	2	1	1	1	3	1	2	1	1	3	1	1	49
1	2	2	2	1	1	3	1	2	2	3	2	2	1	1	3	1	3	2	1	3	1	1	3	50
3	2	2	2	1	1	1	3	1	2	2	3	2	1	1	3	1	1	2	3	2	3	2	1	51
2	2	2	3	2	3	1	1	3	1	2	3	1	1	3	2	1	2	2	2	3	2	1	2	52
2	3	2	3	2	2	2	1	3	1	1	2	2	2	1	3	2	1	2	3	2	3	2	1	53
3	1	2	1	1	2	3	1	2	2	1	2	1	3	1	1	1	3	2	3	2	2	2	3	54
3	2	2	1	2	2	2	3	2	1	1	3	2	2	1	1	3	1	2	1	3	2	1	3	55
1	3	2	2	2	1	2	2	3	1	1	1	3	1	3	2	2	2	3	1	1	2	1	3	56
2	2	3	2	3	2	2	2	1	2	2	3	2	3	2	1	3	2	2	2	1	1	1	3	57
1	2	2	3	2	3	1	3	1	1	3	1	2	1	2	3	1	1	1	3	2	2	1	2	58
2	3	1	3	1	1	2	3	2	1	1	1	3	1	1	2	3	2	2	2	1	2	2	3	59
1	2	3	2	3	1	1	1	3	2	2	1	2	3	1	2	3	2	2	1	1	2	2	3	60
3	2	2	2	1	3	2	1	2	2	1	3	2	2	3	2	2	1	1	3	1	2	2	3	61
3	1	2	2	3	1	2	1	2	2	2	3	1	1	2	3	2	2	2	3	2	2	2	3	62
2	3	1	1	2	2	3	1	1	1	3	2	3	2	1	1	2	3	2	2	3	2	1	2	63
3	1	2	2	3	2	1	2	2	3	2	2	3	1	3	1	1	2	1	3	1	1	2	1	64
1	1	1	2	2	2	3	1	3	1	2	2	2	3	2	3	1	2	1	3	1	3	2	1	65
3	2	1	1	2	2	1	3	1	2	2	2	3	2	2	2	3	2	2	3	2	2	3	2	66
3	2	2	2	3	2	1	2	2	3	2	2	1	3	2	3	1	1	2	1	2	1	3	2	67
1	2	3	2	1	3	2	1	3	2	1	3	1	2	3	2	2	2	1	2	3	1	1	2	68
2	3	2	2	2	1	1	1	3	1	2	3	1	2	2	3	1	1	3	1	1	1	2	3	69
2	3	2	3	1	2	1	1	2	3	1	2	3	2	2	1	2	2	2	3	2	3	2	1	70
1	2	1	3	2	2	3	2	3	1	3	1	1	2	2	2	3	2	1	1	2	2	1	3	71
1	2	1	3	1	2	3	2	1	1	3	1	3	1	1	1	2	2	3	2	3	1	1	1	72
1	3	1	2	2	1	1	3	1	3	1	1	3	2	2	1	1	2	1	3	1	3	2	1	73
3	1	1	3	2	1	1	1	2	2	3	2	3	1	1	2	3	1	1	1	3	1	1	1	74
1	1	2	3	2	1	1	3	1	1	1	3	1	1	3	1	2	2	3	2	2	3	2	1	75
2	2	2	3	1	2	2	2	1	2	3	2	3	2	2	1	2	3	2	2	3	1	3	2	76
3	2	1	2	2	3	1	3	1	1	1	2	2	2	3	1	1	3	1	1	2	3	1	1	77
3	1	1	2	2	3	2	1	2	3	1	1	1	2	3	1	1	2	2	3	2	1	1	3	78
2	1	2	2	3	2	1	3	1	1	3	2	1	1	1	3	2	2	1	3	1	1	3	2	79
2	2	2	1	2	3	2	1	1	2	3	1	2	1	1	3	2	3	2	1	3	2	2	3	80
1	2	1	2	1	3	2	2	3	1	1	1	2	2	3	2	3	1	2	1	3	2	3	2	81
1	2	1	1	3	1	1	1	2	2	1	3	1	3	1	3	2	2	3	2	1	1	1	3	82
3	1	1	2	2	3	2	3	1	1	1	2	3	2	3	1	2	2	3	1	2	1	2	1	83
1	1	1	2	1	1	3	2	1	3	2	2	2	1	1	2	3	1	3	1	3	1	1	3	84
3	1	2	2	1	1	1	3	1	1	3	2	1	1	3	2	3	1	1	2	3	2	2	2	85
2	1	2	3	2	3	2	3	2	2	3	2	2	2	1	3	2	3	2	2	1	2	2	1	86
3	1	3	2	2	1	2	1	2	3	2	1	3	2	2	1	3	1	3	2	2	1	2	1	87
3	1	1	1	3	1	1	1	3	1	1	3	2	3	2	2	1	1	3	2	2	1	1	1	88
2	1	3	2	1	2	2	1	3	2	1	1	3	2	1	2	3	2	3	1	2	2	3	2	89
2	2	3	2	3	2	3	1	2	2	3	1	1	2	1	2	2	3	2	3	1	1	1	2	90
1	2	3	2	3	1	1	1	3	1	3	2	2	1	1	3	2	3	1	2	2	1	1	1	91
3	1	2	2	3	1	1	2	3	1	2	2	3	1	3	1	2	1	2	3	2	1	1	1	92
1	1	3	1	2	3	1	2	1	3	2	2	1	1	3	2	3	2	1	1	3	2	2	1	93
2	1	3	2	2	3	2	2	1	2	2	3	1	3	1	1	2	2	2	1	3	1	1	3	94
2	2	2	1	2	1	3	2	3	1	1	2	2	1	2	3	1	3	2	3	1	1	1	3	95
3	1	2	1	3	1	2	2	2	1	3	1	1	2	3	1	1	2	2	1	1	3	2	3	96
2	2	2	3	1	1	3	1	1	3	1	3	1	2	2	2	3	1	1	1	2	2	3	1	97
1	2	3	1	1	2	1	1	3	1	3	2	2	3	1	2	1	1	1	2	3	2	3	1	98
2	3	2	2	2	1	2	3	2	1	3	2	3	2	1	3	1	2	2	3	1	1	2	2	99
2	2	2	1	1	3	2	3	1	3	2	2	1	2	1	3	1	1	3	2	1	3	2	1	100
3	1	2	2	2	1	2	3	2	3	2	2	2	3	1	1	3	2	2	1	1	3	1	2	101
2	1	3	2	2	1	3	1	3	1	1	1	3	2	3	1	2	1	1	1	3	2	2	1	102
3	2	1	1	2	3	1	2	1	1	2	3	1	1	3	2	3	2	1	2	1	2	1	3	103
1	1	2	3	1	1	3	2	3	2	2	1	3	2	1	2	1	3	1	2	1	3	2	1	104
2	1	1	1	2	2	3	1	3	2	2	2	3	2	2	2	3	1	2	2	3	2	1	3	105
2	1	1	2	3	1	1	3	1	1	2	1	1	3	2	1	2	3	1	3	2	3	2	2	106
1	1	1	2	3	2	1	1	2	1	3	2	3	2	2	3	2	2	1	3	2	2	1	3	107
1	3	1	3	2	2	1	3	2	3	1	1	1	2	3	2	2	3	2	2	1	1	1	2	108
3	1	1	1	2	1	3	1	1	1	2	3	2	1	2	2	3	2	2	2	3	2	3	1	109
1	3	2	2	1	2	1	1	3	2	2	2	3	2	3	1	3	1	1	2	2	1	1	3	110
3	1	3	2	2	2	1	2	1	3	2	2	1	3	1	1	2	1	2	3	2	2	3	2	111
1	3	1	3	2	2	1	2	2	1	3	1	1	3	1	1	3	1	2	2	2	1	1	3	112
3	1	3	2	2	1	1	2	3	1	1	1	2	1	1	3	2	1	2	2	2	3	2	3	113
1	2	3	1	2	3	1	1	2	1	3	2	2	3	1	1	3	2	1	2	1	2	1	3	114
1	2	1	3	1	2	1	2	3	1	3	1	2	3	1	1	1	3	2	2	1	3	2	1	115
2	1	2	3	2	1	1	1	3	1	1	1	3	2	3	1	1	1	3	1	1	3	1	1	116
2	3	1	1	2	3	2	1	3	1	1	1	2	3	1	1	2	3	2	2	3	1	1	1	117
1	1	2	2	3	1	1	2	1	3	2	3	2	3	2	3	1	3	2	2	2	1	1	2	118
1	3	1	2	1	2	2	3	2	2	2	3	1	2	2	1	1	2	3	1	1	3	1	3	119
1	1	1	3	2	2	3	2	1	1	1	3	2	2	3	1	1	3	1	2	1	1	1	3	120
3	2	2	1	1	3	1	3	1	2	2	1	2	3	1	3	1	2	3	2	1	2	2	1	121
1	3	1	1	3	1	2	1	2	1	1	3	1	1	3	1	2	2	3	1	1	2	2	3	122
3	2	1	3	1	1	1	2	2	2	3	1	1	2	2	3	1	2	3	2	3	1	1	1	123
1	1	3	1	3	2	1	3	1	2	2	3	1	2	1	1	3	2	1	2	1	2	3	1	124
2	3	1	2	1	2	1	3	2	1	3	2	3	1	1	3	1	1	1	2	1	1	3	2	125
1	3	1	2	1	1	2	3	1	2	3	1	3	1	1	1	2	3	1	1	3	1	2	1	126
1	2	3	2	3	1	1	1	3	2	1	2	2	2	3	2	3	1	2	1	2	1	3	2	127
1	1	2	1	1	3	1	3	1	1	2	2	3	1	2	1	2	3	1	1	3	1	2	3	128
2	1	1	3	2	3	2	1	2	2	2	1	3	2	1	3	1	1	2	3	1	1	3	2	129
2	1	2	3	2	2	1	3	1	2	2	2	3	2	2	3	1	3	1	2	2	3	1	2	130
1	3	2	2	2	3	2	1	2	3	1	1	3	1	3	1	2	1	3	2	1	2	2	2	131
3	1	3	1	1	1	2	3	2	2	1	2	3	2	1	2	2	2	1	3	2	1	3	2	132
2	1	2	3	2	3	1	3	1	1	2	3	2	3	2	2	2	3	1	2	2	2	1	1	133
3	2	1	2	3	2	2	2	3	2	2	2	1	2	1	3	1	1	2	3	2	1	2	3	134
3	1	3	2	1	2	1	2	1	3	1	1	3	1	1	1	3	1	1	1	2	2	2	3	135
1	2	3	1	3	2	3	1	1	3	2	1	1	1	2	3	2	1	3	2	2	1	2	2	136
2	2	1	1	3	1	1	3	2	3	1	3	2	2	1	2	2	3	2	3	1	2	1	2	137
1	2	3	1	1	1	2	3	1	3	1	1	2	1	2	2	3	2	2	3	2	2	2	3	138
3	1	2	2	1	1	2	3	1	2	2	1	2	3	2	3	1	1	2	2	3	1	2	3	139
3	1	1	1	2	3	2	2	1	1	1	3	1	2	1	2	3	1	1	1	3	2	1	3	140
2	1	2	2	3	2	2	3	1	2	2	2	3	1	2	1	2	2	1	3	2	3	2	3	141
2	2	2	1	2	3	2	2	2	3	2	3	2	1	2	3	2	1	1	3	2	1	3	2	142
1	1	2	2	3	1	1	1	3	1	1	2	2	3	2	3	2	3	1	1	2	2	3	1	143
2	3	1	3	2	2	2	3	1	1	2	2	2	3	2	2	2	3	1	3	2	1	1	2	144
3	1	2	3	2	1	2	1	1	2	3	1	2	3	2	3	2	3	2	1	1	1	2	2	145
1	2	3	2	3	1	3	1	3	1	1	3	1	1	2	2	2	3	2	2	2	1	2	2	146
3	2	3	1	2	1	1	1	3	2	1	2	2	3	2	2	3	1	2	1	3	1	1	1	147
3	1	1	3	2	1	3	1	1	2	1	3	1	1	1	3	2	2	1	1	2	1	3	1	148
2	2	3	2	3	2	1	3	2	2	1	1	3	1	3	2	2	3	2	2	2	1	1	2	149
2	1	3	2	1	3	2	1	1	3	2	2	3	2	2	1	3	1	1	2	1	3	2	2	150
1	1	2	2	2	3	1	1	3	2	1	2	1	1	2	3	1	1	2	3	2	3	2	3	151
2	1	3	1	1	1	2	2	3	2	1	3	2	1	2	2	2	3	1	3	1	3	1	1	152
2	3	2	1	2	1	2	3	2	2	1	1	2	3	1	3	1	2	3	2	2	3	2	1	153
2	1	2	2	2	3	1	2	1	1	3	1	3	1	1	2	3	1	1	3	1	1	3	2	154
2	2	3	1	1	2	1	3	2	3	2	1	1	2	3	1	1	2	1	2	3	1	2	3	155
3	2	1	3	2	2	2	3	2	3	1	1	2	1	3	1	1	2	2	1	3	2	2	2	156
1	1	1	3	1	2	3	1	2	2	3	2	1	1	2	2	2	3	2	3	2	3	1	1	157
3	1	1	3	1	2	2	3	2	2	3	1	3	2	2	1	1	2	1	3	1	2	1	1	158
1	3	1	2	2	1	2	3	2	1	3	2	3	1	2	3	2	1	1	1	2	3	2	2	159
3	1	1	2	2	2	1	3	1	2	3	2	1	3	1	2	1	2	3	1	1	2	3	2	160
3	1	2	1	3	1	1	3	2	3	2	1	2	2	1	1	3	2	1	1	3	2	2	1	161
2	1	2	3	1	1	2	2	1	2	3	1	3	1	1	3	1	1	2	1	3	1	3	2	162
2	2	2	3	2	2	1	2	3	1	1	3	2	3	1	2	2	2	3	2	2	2	3	2	163
3	2	1	1	1	3	1	2	2	3	2	3	2	2	1	2	1	2	3	1	1	1	2	3	164
2	2	3	2	3	1	2	1	3	2	1	3	2	2	1	3	1	2	1	2	2	2	3	2	165
3	1	1	2	2	1	1	3	1	2	1	1	1	3	1	1	3	1	3	1	1	3	2	1	166
3	1	2	2	3	2	1	3	1	1	2	3	1	1	2	2	2	3	2	1	3	2	1	2	167
1	1	1	2	1	1	3	1	3	1	3	1	3	1	1	2	3	1	2	2	2	1	3	2	168
1	1	2	2	1	2	3	2	3	1	1	2	1	3	1	2	2	3	2	2	3	1	1	3	169
2	2	1	1	3	1	2	2	2	1	2	3	2	3	1	2	1	3	2	1	3	1	3	2	170
2	2	1	1	1	3	1	2	1	3	2	3	2	2	2	3	2	2	3	2	3	2	2	1	171
2	1	2	2	3	1	2	2	2	1	2	3	1	1	3	1	3	2	1	2	1	3	2	3	172
1	1	1	2	2	2	3	1	2	3	1	3	2	1	3	2	2	2	1	1	3	1	3	1	173
1	2	1	1	1	3	2	2	3	2	2	2	3	1	2	3	2	2	2	3	1	1	2	3	174
3	1	2	2	3	2	3	1	2	3	1	1	2	1	1	2	3	2	2	1	2	2	3	1	175
3	1	2	3	1	1	3	1	1	1	2	1	2	3	1	2	1	2	3	1	1	2	1	3	176
2	2	1	1	1	3	2	2	1	2	2	3	1	1	3	2	3	1	1	3	2	2	3	1	177
2	2	3	2	1	1	3	1	1	1	2	1	3	1	3	1	2	2	2	3	2	3	2	2	178
3	1	3	1	2	2	3	1	3	2	2	2	1	1	3	2	1	2	2	1	3	1	2	2	179
1	3	2	3	1	2	1	1	2	1	3	1	1	2	3	1	2	1	1	1	2	3	2	3	180
3	1	2	1	1	2	1	3	2	3	1	1	2	2	2	3	1	3	2	2	3	2	1	2	181
1	3	1	2	1	2	2	2	3	2	1	3	2	1	3	1	1	1	3	2	1	2	3	2	182
3	2	2	1	2	3	1	1	2	3	2	2	3	1	1	2	2	2	3	1	1	2	3	2	183
1	2	3	1	1	1	3	1	2	2	2	1	3	2	2	3	2	3	1	3	1	2	1	2	184
1	1	1	2	1	3	1	3	1	1	3	2	2	1	2	3	1	2	3	2	3	1	2	1	185
2	2	1	3	2	3	1	3	1	1	1	2	3	2	2	2	1	1	2	3	2	3	1	2	186
2	3	1	1	3	1	1	2	1	2	3	2	3	1	1	1	2	2	1	3	2	2	2	3	187
3	2	2	2	3	1	2	1	3	2	2	2	1	1	2	3	1	3	2	1	2	2	3	1	188
3	2	2	3	2	1	1	3	2	1	1	2	3	1	2	1	1	1	3	2	1	2	3	1	189
2	1	1	3	1	3	2	1	3	2	1	1	2	2	3	2	2	3	2	2	2	1	3	1	190
2	2	2	3	1	3	1	3	1	3	2	1	2	3	2	1	2	3	1	2	2	1	2	2	191
1	2	2	3	1	2	2	3	2	3	1	1	2	2	1	3	1	2	1	3	1	1	3	1	192
3	1	2	2	1	3	2	1	2	2	2	1	3	2	1	3	2	1	1	2	1	3	1	3	193
2	1	2	3	2	1	2	2	1	3	1	3	1	2	1	2	2	3	1	1	1	3	2	3	194
2	1	2	3	2	3	1	1	1	3	2	1	1	2	3	1	2	1	1	1	2	3	1	3	195
3	2	1	1	2	2	1	3	2	1	1	2	3	1	2	2	2	3	1	1	2	3	1	3	196
3	2	2	2	1	2	2	3	2	1	1	1	3	1	2	3	2	1	1	3	2	3	1	1	197
2	1	3	2	1	3	1	1	2	2	3	2	2	3	2	2	1	1	1	3	1	1	2	3	198
2	1	2	2	3	2	2	1	3	2	2	1	2	3	2	1	3	2	3	2	3	2	1	1	199
3	1	3	2	3	1	1	1	3	2	2	1	2	1	2	3	1	1	1	3	2	1	2	1	200
1	2	1	2	1	3	1	1	3	2	2	3	1	2	3	1	3	2	2	2	1	2	3	1	201
2	2	2	1	3	1	1	3	2	1	1	3	1	1	2	1	1	3	2	3	1	3	2	1	202
2	3	2	3	2	1	2	1	1	3	1	2	1	2	2	2	3	2	1	1	3	1	1	3	203
2	1	3	1	1	3	1	3	2	2	3	2	1	2	2	3	2	2	1	2	1	1	3	2	204
3	2	3	2	2	1	2	2	1	3	2	2	2	1	1	3	2	2	1	3	1	3	2	1	205
1	1	2	1	2	1	3	2	3	1	2	3	2	3	1	1	1	2	2	3	1	1	2	3	206
2	2	1	3	1	3	1	1	2	1	3	1	3	2	3	1	2	2	1	2	1	3	2	2	207
3	1	1	3	2	3	1	3	2	2	1	1	2	3	1	2	2	2	3	2	1	1	1	2	208
1	1	2	3	2	1	1	1	3	2	1	1	1	3	1	1	1	3	2	3	1	2	3	1	209
3	2	2	1	3	2	2	1	2	3	1	2	3	1	1	2	1	2	2	3	2	3	2	1	210
1	1	1	2	3	1	3	2	2	1	3	1	3	2	1	3	1	1	2	2	1	2	3	2	211
3	1	2	1	2	1	3	1	1	3	1	2	2	1	3	2	2	1	3	2	3	1	2	1	212
1	2	1	3	2	2	2	3	2	2	3	1	3	1	2	2	2	1	2	3	1	3	2	1	213
2	1	3	1	1	2	1	3	2	2	1	3	2	1	3	2	1	1	3	1	3	2	1	2	214
3	1	1	2	2	2	3	2	1	2	2	3	2	3	1	1	3	2	2	2	1	3	2	1	215
3	2	1	3	2	1	1	3	1	1	3	1	3	1	1	2	2	1	3	1	2	2	1	1	216
1	1	2	3	2	3	2	2	1	2	3	2	1	2	3	2	1	1	1	2	1	3	2	3	217
3	1	1	2	2	1	3	2	2	1	3	1	3	2	1	1	1	2	2	3	2	2	2	3	218
3	1	1	1	2	2	3	1	1	3	1	2	1	3	2	1	1	3	1	1	1	2	3	1	219
3	2	3	2	1	2	2	1	2	3	2	3	1	2	2	2	1	2	3	1	2	1	3	1	220
2	1	2	2	1	2	3	1	3	1	1	1	3	2	2	3	1	1	2	1	3	2	1	3	221
2	1	2	3	2	1	2	2	3	2	1	2	2	3	1	3	2	1	3	1	2	3	1	1	222
3	2	3	1	2	2	3	1	1	2	1	3	2	1	3	1	2	2	3	2	2	2	1	1	223
1	3	2	1	1	3	2	2	3	2	2	2	3	1	2	2	3	1	1	1	2	2	2	3	224
3	1	1	3	2	2	2	3	1	2	2	2	1	1	3	2	2	2	1	1	3	1	1	3	225
3	1	3	1	1	3	1	2	1	1	1	2	3	1	2	1	2	2	3	2	2	1	2	3	226
1	2	3	1	2	3	1	3	2	2	3	2	2	1	1	2	1	3	2	2	1	3	2	2	227
2	1	2	3	1	2	1	2	2	2	3	1	1	3	1	3	2	3	2	2	1	1	3	1	228
3	1	3	1	2	3	1	2	2	1	1	1	3	2	3	1	2	2	2	1	2	3	1	1	229
1	2	1	3	2	2	1	1	3	1	3	2	3	1	2	3	1	3	1	1	2	1	1	1	230
2	2	2	1	2	2	3	2	2	1	3	1	2	1	1	1	3	1	3	2	2	3	1	3	231
1	3	1	1	2	1	2	2	3	1	2	1	3	2	2	3	1	1	3	2	2	3	1	1	232
2	1	3	2	3	2	1	1	1	3	2	3	2	1	3	1	2	2	3	2	1	1	1	2	233
1	3	2	1	3	2	3	1	2	1	2	3	1	2	2	2	3	1	1	2	1	2	2	3	234
2	3	2	1	2	2	3	1	1	2	2	1	3	1	1	2	1	3	2	3	1	3	1	1	235
2	3	1	2	1	2	3	1	3	1	2	1	3	1	1	3	2	2	2	1	1	2	3	2	236
3	1	1	3	1	1	3	2	1	1	3	2	1	2	1	1	1	3	2	1	1	1	2	3	237
2	2	2	1	1	3	2	3	2	3	1	2	1	1	3	1	1	1	3	1	2	1	3	1	238
2	1	2	2	3	2	2	3	1	1	2	3	2	3	2	2	2	1	1	1	3	1	3	1	239
3	1	1	2	1	1	2	3	1	2	3	1	3	1	2	3	1	2	2	1	2	2	3	1	240
2	1	3	1	3	1	1	1	3	1	3	1	3	1	1	2	2	3	2	1	2	2	1	1	241
1	2	3	2	1	2	1	1	2	3	1	3	1	2	1	2	3	2	2	2	3	2	3	1	242
1	1	2	1	3	1	2	1	1	3	1	2	2	3	1	2	2	3	2	3	2	2	2	3	243
2	2	2	3	1	2	3	1	2	1	1	2	1	3	1	1	3	1	3	1	1	2	3	1	244
1	3	1	2	3	1	1	2	1	1	3	2	2	3	2	3	1	1	2	3	2	2	2	1	245
1	3	1	2	3	1	1	1	3	1	1	1	3	2	3	2	1	3	1	1	2	1	2	2	246
2	3	2	2	1	1	1	2	3	2	1	2	3	2	1	3	2	1	1	2	2	3	1	3	247
2	1	3	2	1	3	2	3	2	3	1	1	3	2	2	1	2	2	2	3	2	2	1	2	248
1	3	2	3	1	1	2	3	2	2	2	3	2	1	1	1	3	1	3	2	2	2	1	1	249
3	1	2	1	1	1	2	3	1	3	1	1	2	2	3	1	3	2	1	1	2	2	3	2	250
2	3	1	2	3	1	3	1	1	1	2	2	3	2	2	2	1	1	3	2	3	2	2	2	251
1	1	1	2	1	1	3	2	1	3	2	3	2	3	1	3	2	1	1	2	1	3	2	1	252
2	1	2	3	1	1	1	2	1	2	3	2	3	1	2	1	3	2	1	1	3	1	3	1	253
1	2	2	3	2	1	1	3	1	3	2	3	1	2	2	1	2	1	3	1	2	3	1	2	254
1	3	1	3	2	1	1	3	1	1	2	3	1	1	1	3	1	3	1	2	1	1	2	1	255
2	1	1	3	2	1	1	3	2	1	3	1	2	3	2	2	1	1	1	3	1	3	1	2	256
1	1	1	2	1	3	1	1	1	3	1	1	2	2	3	2	1	3	1	3	2	1	3	2	257
1	2	1	3	1	2	2	2	1	1	3	2	3	1	1	3	1	3	1	3	2	2	1	2	258
3	1	1	2	3	2	2	2	3	2	1	1	1	2	3	2	1	2	1	3	1	2	1	3	259
1	1	1	2	1	3	1	1	2	3	1	3	2	1	3	2	3	1	1	1	2	1	2	3	260
2	2	3	1	1	2	2	1	2	3	2	1	3	1	3	1	1	1	3	2	1	1	1	3	261
2	1	3	2	1	1	1	2	2	3	1	3	1	3	2	1	3	2	2	3	1	1	2	2	262
2	3	2	1	1	1	3	2	3	2	2	2	1	2	1	3	2	3	2	3	2	1	1	2	263
1	2	1	2	3	1	2	2	2	3	1	3	1	2	3	1	3	1	1	2	3	2	1	1	264
1	1	2	1	2	2	3	1	2	1	2	3	2	3	2	2	3	2	3	1	1	3	2	1	265
1	3	2	3	1	3	1	2	2	1	2	3	1	3	2	1	2	2	3	1	2	2	2	1	266
2	2	3	2	1	2	2	2	1	3	1	2	1	3	2	3	1	3	1	2	2	1	2	3	267
1	2	1	3	1	1	1	2	3	1	1	1	3	1	2	1	3	1	2	1	3	1	1	3	268
3	1	2	2	3	2	1	2	1	2	3	2	1	1	1	3	2	1	3	2	2	2	1	3	269
2	1	2	3	1	1	2	3	2	2	1	2	2	3	2	3	2	3	2	2	3	1	2	2	270
3	1	2	1	2	2	1	3	2	1	3	1	3	2	1	1	3	2	1	2	1	2	2	3	271
2	3	1	3	1	2	3	1	1	2	2	2	3	2	3	2	2	1	2	3	1	2	1	2	272
2	1	2	3	1	1	2	3	1	1	3	2	1	1	1	3	1	3	1	2	3	2	1	1	273
3	1	3	2	3	1	1	2	2	2	3	2	2	3	2	1	1	2	2	2	3	2	2	2	274
1	3	1	1	1	2	2	3	2	1	3	1	3	2	2	1	1	2	2	3	2	3	2	1	275
3	2	3	2	2	1	1	2	3	1	1	1	3	2	2	3	2	3	1	1	2	1	1	2	276
2	3	2	3	1	2	2	2	3	2	2	1	1	3	1	1	3	1	2	2	1	1	2	3	277
1	3	2	1	3	2	1	2	2	3	2	1	1	1	3	2	1	2	1	1	1	3	1	3	278
2	3	1	2	2	3	2	2	3	2	1	2	1	3	2	2	1	2	2	3	2	3	2	1	279
3	1	2	2	3	2	1	3	2	2	2	1	1	2	3	2	2	1	1	3	1	1	2	3	280
1	2	3	1	1	1	2	1	1	3	1	1	1	2	2	3	1	3	2	1	3	1	3	1	281
2	1	2	3	1	2	3	1	2	1	2	2	2	3	2	2	3	2	1	2	3	2	3	2	282
2	2	2	1	3	1	3	2	2	2	3	1	2	2	1	3	2	1	2	3	2	2	2	3	283
1	1	2	1	1	3	1	3	1	2	2	3	2	3	1	2	3	1	3	1	1	1	2	1	284
1	1	2	3	1	1	2	1	3	1	1	2	1	3	1	3	1	1	2	3	2	1	3	1	285
3	2	1	3	2	1	3	2	1	1	2	2	2	3	1	1	2	3	2	2	2	3	1	1	286
1	3	2	3	1	3	2	1	1	2	2	3	1	2	2	3	1	2	2	3	2	2	1	1	287
3	1	1	2	1	1	2	3	2	2	2	1	3	2	3	2	3	2	2	2	3	1	1	1	288
1	2	1	2	3	1	1	1	3	2	1	3	1	3	1	1	1	3	2	3	2	2	1	2	289
2	3	1	3	2	2	1	2	2	3	2	1	2	2	2	1	3	2	2	2	3	1	1	3	290
2	1	3	2	2	3	1	3	2	2	2	1	1	1	3	2	2	3	1	1	1	3	1	1	291
2	1	1	1	3	1	3	2	3	1	2	3	2	1	1	1	2	1	3	1	1	3	2	2	292
2	3	2	1	3	2	3	2	2	2	1	3	1	3	2	1	1	3	2	2	1	2	2	1	293
1	3	1	3	1	2	2	1	1	2	3	2	3	2	2	3	1	1	1	3	1	2	2	1	294
3	2	1	1	2	1	1	3	2	2	3	2	3	1	1	1	3	1	1	3	1	2	2	1	295
3	1	3	1	2	3	2	2	1	2	1	3	1	2	1	1	2	3	1	1	1	3	1	1	296
2	2	2	1	3	2	2	3	1	2	2	3	2	2	3	1	1	2	1	3	1	3	2	1	297
1	2	2	1	2	2	3	1	1	1	3	2	1	3	1	2	3	2	2	1	3	1	2	3	298
2	2	2	1	2	3	2	3	2	3	1	2	2	3	1	3	2	3	2	2	2	1	1	2	299
2	1	2	2	2	1	3	2	2	1	3	1	2	1	3	1	2	1	3	1	3	1	3	2	300
1	2	3	2	3	2	2	2	1	2	3	2	3	1	1	1	3	1	2	2	2	3	2	1	301
1	2	1	3	2	1	1	2	2	1	3	1	1	3	1	3	1	1	3	1	1	2	3	2	302
2	1	2	3	1	3	2	3	1	2	2	1	3	1	1	2	2	3	2	1	2	2	2	3	303
2	2	1	1	2	3	2	1	2	2	3	2	2	2	1	1	1	3	1	3	2	3	2	3	304
1	2	1	3	1	3	1	1	2	2	1	1	3	1	1	2	2	3	2	2	2	3	1	3	305
3	2	2	1	2	1	1	3	2	1	3	1	1	1	2	3	2	1	2	1	3	1	1	3	306
1	3	2	1	1	2	2	1	3	2	2	2	3	1	1	1	2	3	2	3	2	1	3	2	307
3	1	1	1	3	1	2	2	1	2	3	1	2	2	3	2	1	1	1	3	2	3	1	2	308
3	2	1	1	3	1	2	2	1	3	1	1	3	2	2	1	1	2	3	1	1	3	1	1	309
3	1	3	1	1	2	3	2	2	3	1	1	2	1	1	3	1	1	3	2	1	1	2	2	310
2	2	1	1	3	1	3	2	3	2	2	2	3	1	1	2	1	3	2	3	2	2	2	1	311
1	2	1	1	1	3	1	1	1	3	1	3	2	1	2	3	1	3	1	2	2	1	2	3	312
1	3	2	2	1	2	2	3	1	2	2	3	1	1	3	1	2	3	1	3	1	1	1	2	313
3	2	2	2	3	2	3	2	2	2	3	2	1	2	1	1	3	2	2	3	2	2	1	1	314
2	2	3	2	1	2	3	2	3	1	3	2	2	2	1	3	1	2	2	1	1	2	3	1	315
2	1	3	2	2	1	1	1	3	2	1	2	1	3	2	2	3	2	2	2	3	1	3	2	316
1	1	1	2	2	2	3	2	3	2	2	3	1	3	1	2	2	2	3	2	1	2	1	3	317
2	1	2	2	1	3	2	3	2	2	1	2	3	1	2	1	1	1	3	1	3	1	1	3	318
2	1	2	1	1	3	1	1	3	2	1	1	2	2	2	3	1	3	1	1	3	1	3	2	319
2	1	1	3	2	2	3	1	3	1	2	3	2	2	2	3	2	2	2	3	1	2	1	1	320
3	2	3	2	1	3	1	2	2	2	1	2	3	1	1	2	2	3	1	3	2	1	1	2	321
2	1	2	1	3	1	3	1	1	3	2	3	2	2	2	1	3	2	2	3	2	1	2	1	322
1	2	1	1	1	3	1	1	3	1	1	2	1	3	2	2	3	2	2	3	2	3	2	1	323
1	3	1	2	2	3	1	1	1	2	1	3	1	2	2	1	3	1	1	1	3	2	2	3	324
3	2	2	3	2	2	1	2	1	1	3	1	1	1	2	1	3	2	2	2	3	2	2	3	325
1	3	1	1	1	2	1	3	1	3	2	1	1	3	1	3	2	3	2	2	2	1	1	1	326
1	3	1	3	1	2	1	3	2	1	3	2	1	1	1	2	1	3	2	2	1	2	2	3	327
1	1	1	2	3	1	2	2	3	2	3	2	1	1	3	2	2	1	2	3	2	1	2	3	328
1	1	3	1	1	3	2	1	1	3	1	3	1	3	1	1	1	2	2	2	3	1	1	2	329
3	2	3	2	3	2	1	2	2	2	1	3	2	2	3	1	2	1	1	2	2	3	1	2	330
1	2	2	3	2	2	3	2	2	3	2	2	3	1	3	1	1	1	2	3	2	1	2	2	331
1	3	1	2	1	1	3	2	2	1	1	1	3	2	1	1	1	3	1	3	1	1	2	3	332
2	1	3	2	2	3	1	1	3	2	2	1	3	2	2	2	1	1	3	2	3	2	2	1	333
1	3	2	1	1	3	1	1	2	3	2	1	1	2	1	2	3	1	2	3	1	2	1	3	334
1	2	3	1	3	1	2	2	3	1	1	1	3	1	2	2	2	1	2	3	1	1	2	3	335
2	3	1	2	2	3	1	1	2	2	1	3	1	3	1	3	1	1	2	3	2	1	2	1	336
1	3	2	2	1	3	2	1	1	3	1	3	1	1	2	1	2	1	3	2	3	1	1	2	337
1	2	2	1	1	3	1	2	2	3	2	1	2	1	3	2	2	1	3	2	3	1	2	3	338
3	1	3	1	2	1	1	1	3	1	1	2	2	3	1	1	1	2	1	3	1	1	3	1	339
1	3	1	3	2	1	1	1	2	3	2	2	1	1	3	1	1	1	3	1	1	3	2	2	340
1	1	1	3	2	2	2	3	2	2	1	2	3	2	3	2	3	1	1	3	1	1	2	2	341
1	2	2	3	2	3	2	2	2	1	1	3	1	1	1	2	1	2	3	1	2	3	1	3	342
2	1	2	2	3	1	1	1	2	3	1	3	1	2	3	2	1	2	3	2	1	3	2	2	343
1	2	2	2	3	2	3	2	3	1	2	3	2	2	2	3	1	1	1	2	1	2	3	1	344
2	1	1	3	1	2	1	1	2	1	3	2	3	1	3	1	3	1	1	1	2	2	3	1	345
1	2	2	2	1	2	3	1	2	2	1	3	2	3	2	1	1	3	2	3	2	2	3	2	346
3	1	2	2	1	1	3	1	1	2	1	1	1	3	2	3	2	3	1	1	3	1	1	2	347
3	2	1	1	2	2	3	1	2	3	1	1	3	1	3	2	2	1	3	2	2	2	1	2	348
2	3	2	3	2	2	1	2	3	2	2	1	2	1	1	3	1	1	3	2	3	1	2	1	349
1	3	1	3	1	1	1	2	2	3	1	1	2	2	2	1	3	1	1	1	2	3	2	3	350
2	2	1	2	2	3	1	1	2	3	2	3	1	3	1	1	1	3	2	1	2	2	2	3	351
2	3	2	2	1	1	2	3	1	3	1	1	3	1	2	1	1	2	3	1	2	1	3	2	352
3	1	1	1	3	2	1	2	2	2	3	2	2	3	1	2	2	1	2	2	3	2	2	3	353
2	1	3	2	2	2	1	2	3	2	1	3	2	2	1	1	2	2	3	2	2	3	1	3	354
3	2	2	3	1	1	1	3	1	2	1	3	2	2	2	3	1	2	1	2	3	2	1	2	355
2	2	1	3	1	1	3	1	2	1	3	1	2	2	1	2	2	3	1	3	1	1	1	3	356
1	1	2	1	1	2	3	2	2	3	2	3	1	1	1	2	1	3	1	2	3	2	3	1	357
1	3	2	1	1	3	1	1	1	3	2	2	2	1	3	2	2	2	1	3	2	2	1	3	358
2	1	3	2	2	2	1	1	2	3	1	3	1	2	3	2	2	2	3	1	2	1	2	3	359
2	2	1	1	1	3	1	2	3	2	2	1	1	1	3	1	1	2	3	1	3	2	3	1	360
1	1	1	3	2	3	2	3	2	1	2	1	2	3	2	2	1	3	1	1	1	3	2	1	361
1	2	2	1	1	3	2	2	1	2	3	2	3	2	2	2	1	2	3	2	3	2	2	3	362
2	2	2	3	1	1	3	1	1	3	2	3	2	2	2	3	2	1	2	2	1	2	3	2	363
2	3	2	2	1	1	3	1	1	3	2	2	2	1	3	2	2	1	1	1	3	2	2	3	364
2	2	2	1	1	3	2	1	2	1	1	3	1	2	2	3	2	3	2	3	1	3	1	2	365
1	3	1	2	1	2	2	2	3	1	2	1	3	1	2	1	3	1	1	3	1	1	1	3	366
1	2	2	2	1	3	1	3	2	2	3	2	1	1	3	1	1	3	1	2	1	2	2	3	367
3	1	3	1	1	1	2	2	3	2	1	1	2	2	3	2	2	1	3	1	3	2	1	2	368
3	1	1	3	2	1	2	1	2	3	2	2	1	1	3	1	2	3	2	1	1	2	1	3	369
1	1	2	1	2	2	3	1	1	3	1	2	3	2	1	3	2	3	1	3	2	2	1	2	370
3	1	3	2	2	2	1	3	1	1	1	2	3	1	2	1	1	1	3	1	1	2	2	3	371
2	1	1	3	1	1	1	2	3	1	3	2	2	1	2	1	2	3	2	2	3	1	3	1	372
2	2	3	1	2	1	2	1	1	3	1	1	3	2	2	3	2	3	1	2	1	1	3	2	373
1	1	3	2	3	2	2	2	1	1	2	3	2	1	1	3	1	3	1	1	2	3	1	1	374
3	2	2	3	2	3	1	3	1	1	2	2	1	3	1	1	1	2	1	3	2	1	2	1	375
2	2	2	1	3	2	2	2	3	1	2	3	2	3	2	2	2	1	2	3	1	3	1	2	376
3	2	1	1	2	2	3	1	1	1	3	2	1	2	3	1	3	2	1	3	2	1	1	2	377
2	1	3	2	2	3	1	1	2	1	1	3	1	2	2	3	1	3	1	3	1	1	1	2	378
2	2	1	1	3	2	3	1	1	3	2	3	2	2	3	2	2	2	1	2	2	3	1	1	379
1	2	2	3	1	2	2	2	3	2	2	3	1	1	1	2	1	1	3	2	3	2	2	3	380
2	3	1	1	2	2	3	2	2	3	1	2	1	1	3	2	2	1	2	3	1	1	3	1	381
3	2	2	2	3	2	2	1	2	2	3	1	3	2	1	1	3	2	2	3	1	1	2	2	382
2	3	1	2	2	2	1	3	2	1	2	3	2	1	2	2	1	3	1	3	2	2	3	1	383
2	1	1	1	2	1	3	1	3	1	2	3	1	3	1	1	2	1	1	3	1	1	1	3	384
1	3	1	1	2	3	2	2	1	2	1	2	3	2	1	3	1	3	1	1	1	2	2	3	385
1	2	2	2	1	2	3	2	1	3	2	2	3	1	3	1	3	2	3	1	2	1	1	1	386
3	2	1	1	1	3	1	2	1	3	2	2	2	3	1	3	2	1	1	2	2	2	3	1	387
3	1	1	1	2	1	3	2	1	2	1	1	2	3	2	2	1	1	3	2	3	1	3	1	388
1	2	2	3	2	1	2	1	2	2	3	2	3	2	2	3	1	1	3	1	1	1	3	2	389
3	1	3	2	2	1	1	3	2	3	2	1	1	1	2	3	1	1	1	2	3	2	1	1	390
1	2	1	3	1	2	2	3	2	3	2	3	1	1	1	3	1	1	1	3	1	1	2	2	391
2	2	1	1	2	1	3	1	1	3	2	2	2	3	2	1	3	2	1	2	3	1	2	3	392
2	2	3	2	1	2	3	2	3	1	3	1	1	2	1	1	1	3	2	2	2	1	3	2	393
3	2	3	1	2	2	1	3	1	2	1	2	3	1	2	3	1	2	1	2	3	1	1	2	394
2	3	1	1	3	1	1	3	1	1	2	2	2	1	3	1	2	2	2	3	2	1	1	3	395
2	3	2	1	2	3	1	2	2	1	2	2	3	1	2	2	1	3	2	3	2	3	2	2	396
2	3	2	3	1	1	1	3	1	3	1	1	2	3	1	2	1	3	1	2	1	2	2	2	397
1	1	2	2	3	1	1	1	2	3	1	3	2	3	2	3	2	2	2	1	1	3	1	1	398
1	2	2	1	2	1	3	1	3	2	2	1	3	2	2	2	1	3	1	1	2	3	1	3	399
1	1	1	3	1	2	1	3	1	1	1	2	2	3	1	3	2	3	2	1	2	3	1	2	400
3	2	1	3	2	2	2	3	2	2	1	1	2	3	2	2	3	2	1	2	1	1	2	3	401
1	3	1	3	1	2	1	2	2	1	3	1	1	2	3	2	1	1	3	1	1	2	1	3	402
1	3	1	1	3	2	2	2	3	1	1	1	2	1	2	3	1	2	1	3	1	1	2	3	403
2	1	3	1	1	2	3	2	1	1	1	3	2	2	2	1	3	2	1	2	1	3	1	3	404
1	3	2	1	3	1	2	3	2	1	2	3	2	2	1	1	2	3	2	3	1	1	2	1	405
2	3	1	1	1	3	2	3	1	1	1	2	1	2	3	1	1	1	2	3	2	2	3	2	406
1	2	1	3	2	1	2	1	2	2	3	1	3	2	2	2	3	2	1	2	3	1	1	3	407
3	1	1	3	1	1	1	2	3	2	2	2	3	2	1	3	1	1	2	1	1	3	2	1	408
1	1	2	3	1	3	2	1	2	2	3	1	1	3	1	1	1	2	3	2	1	2	1	3	409
3	2	3	1	2	1	3	1	1	2	2	2	3	2	3	2	2	2	1	1	2	3	1	1	410
2	3	2	1	3	2	1	2	3	1	1	3	1	1	2	1	1	2	3	1	1	1	2	3	411
1	2	1	3	1	1	3	2	2	1	1	2	3	1	2	1	1	2	2	3	2	3	2	3	412
3	2	3	1	2	2	3	2	1	1	3	2	1	1	3	2	1	1	1	3	1	2	1	1	413
2	1	2	3	2	1	3	2	2	2	3	2	3	2	2	1	2	2	2	3	1	1	3	1	414
2	3	1	3	2	1	1	3	2	2	2	3	2	1	2	3	2	2	2	1	1	3	2	1	415
2	1	1	1	2	3	2	1	2	3	1	3	2	3	2	3	2	1	1	1	3	1	1	1	416
3	2	1	1	3	1	3	2	1	2	2	3	1	1	1	2	2	1	3	2	1	1	3	1	417
3	2	2	3	1	3	2	3	2	1	1	1	3	1	2	2	1	2	2	3	1	2	1	1	418
1	3	2	1	2	3	1	3	2	2	1	2	2	1	3	1	2	1	1	1	3	2	3	1	419
1	2	2	2	3	2	2	1	2	1	3	1	3	2	2	3	2	3	2	2	3	2	1	2	420
2	1	1	2	2	1	3	2	1	3	2	3	2	3	2	2	3	1	1	1	2	2	2	3	421
2	3	2	1	2	2	3	1	3	1	2	2	3	2	2	1	2	2	3	2	1	2	2	3	422
3	2	2	1	2	2	1	3	1	1	3	1	3	1	2	1	1	2	2	3	1	3	2	2	423
2	2	3	1	3	2	2	3	2	3	1	2	2	1	1	3	2	1	3	2	1	2	1	2	424
3	1	2	1	3	2	1	2	1	1	2	3	1	2	2	3	1	1	3	2	1	1	2	3	425
3	2	3	1	1	1	3	1	2	1	2	2	2	3	1	3	1	3	1	2	1	1	1	2	426
1	3	2	2	1	2	3	1	2	2	2	3	1	1	3	1	1	1	2	2	3	2	2	3	427
3	2	1	1	3	2	1	2	2	2	3	1	1	2	2	2	3	1	2	3	1	3	2	2	428
2	1	1	2	1	3	2	3	2	2	1	2	1	1	3	2	3	1	1	1	3	1	3	2	429
1	1	1	2	3	1	1	2	2	3	1	2	3	2	3	2	1	2	1	2	3	1	1	3	430
1	3	1	1	1	3	2	3	1	3	2	2	3	2	2	1	1	3	2	1	2	2	2	1	431
2	2	2	1	2	3	2	3	2	3	1	1	2	2	3	2	3	2	1	2	1	2	1	3	432
3	2	1	1	2	1	2	3	1	2	1	3	1	1	1	2	3	2	1	1	1	3	1	3	433
3	1	3	1	1	2	2	3	2	2	2	1	1	1	3	1	2	1	3	2	2	3	2	1	434
3	1	1	2	2	2	3	2	2	1	1	3	1	1	2	3	1	3	2	2	2	3	1	2	435
1	2	1	3	2	3	1	2	3	1	2	2	1	1	1	3	1	3	1	1	2	2	2	3	436
1	2	1	3	1	2	3	2	2	2	1	3	2	2	3	1	3	1	2	2	1	2	2	3	437
1	1	3	1	3	2	3	2	1	1	1	2	1	3	1	1	1	3	2	3	1	2	1	2	438
2	3	2	3	2	1	2	2	3	1	2	2	3	2	2	3	1	3	1	2	1	1	1	2	439
2	1	3	2	1	2	1	3	2	3	1	3	1	1	1	3	1	3	2	2	1	1	1	2	440
1	1	1	3	1	2	1	1	3	1	1	1	3	1	3	1	2	3	1	2	3	2	2	2	441
3	1	1	3	2	2	1	2	2	3	1	1	1	2	1	3	1	3	1	1	3	2	1	2	442
1	2	3	2	1	2	3	2	1	2	1	3	1	1	1	3	1	3	2	1	1	1	2	3	443
3	1	2	3	2	2	2	3	2	1	1	1	3	1	2	2	3	1	1	1	2	2	3	1	444
1	1	2	2	2	1	3	1	3	1	3	2	1	2	2	2	3	2	3	2	2	3	2	1	445
1	1	2	2	2	3	2	2	2	3	1	1	1	3	1	1	1	3	2	1	1	3	2	3	446
1	1	1	3	1	3	2	1	3	2	3	2	2	1	2	2	3	2	2	1	3	1	2	1	447
3	2	1	2	3	2	2	3	2	1	2	1	2	3	2	2	3	2	2	3	1	2	1	2	448
3	2	1	3	1	1	2	2	2	3	2	2	3	1	3	2	1	2	2	2	3	2	1	1	449
1	2	3	1	1	2	2	2	1	3	2	2	1	3	2	3	2	1	1	3	1	1	1	3	450
1	2	3	1	2	1	1	3	1	1	1	2	3	2	2	3	1	2	3	1	1	3	2	1	451
2	2	3	1	2	3	1	2	3	1	1	3	1	2	1	1	2	3	2	1	3	1	2	1	452
1	3	1	2	3	1	2	1	2	3	1	2	1	2	1	3	1	2	2	1	3	1	2	3	453
2	2	3	1	1	1	3	2	2	1	3	1	1	1	3	1	2	1	3	1	2	3	2	2	454
3	2	2	2	1	1	2	3	2	2	1	3	2	2	1	3	1	1	1	3	2	1	1	3	455
3	1	3	1	2	2	2	1	1	3	2	2	2	3	1	1	3	2	3	1	1	1	2	1	456
2	2	2	3	2	2	1	3	2	1	3	2	2	3	2	2	1	2	1	1	3	1	3	1	457
2	1	2	3	1	3	1	1	2	1	3	2	2	2	3	2	2	1	3	2	3	1	1	2	458
2	2	3	1	1	1	3	2	2	2	1	1	1	3	1	1	3	1	3	1	2	1	1	3	459
1	1	3	2	3	1	3	2	2	3	1	1	1	2	3	1	1	1	2	1	2	3	2	2	460
3	2	2	1	3	1	1	1	2	3	1	1	1	2	3	1	3	2	1	3	2	2	1	2	461
2	1	1	3	2	1	2	2	3	2	1	2	2	2	3	2	3	2	3	2	3	2	1	2	462
2	3	2	1	2	2	1	3	2	1	1	1	3	1	1	3	1	3	1	3	1	1	2	1	463
3	1	3	1	1	3	1	3	1	1	1	2	1	1	3	2	2	3	1	1	1	2	1	1	464
3	2	1	1	1	3	2	1	3	1	1	1	2	1	3	1	1	2	2	3	1	3	2	2	465
3	2	3	2	3	2	2	1	2	2	2	3	2	2	2	3	2	1	1	1	3	2	1	2	466
2	2	2	3	1	2	3	2	1	2	3	1	1	2	1	2	1	3	2	1	2	3	1	3	467
1	1	3	1	2	2	3	2	3	2	3	1	1	2	1	3	2	2	3	1	1	1	2	2	468
2	1	2	1	1	1	3	2	2	2	3	1	1	3	1	2	3	1	3	2	3	1	2	1	469
1	3	1	2	1	1	1	3	1	3	1	2	2	2	1	1	3	1	2	3	2	1	2	3	470
3	1	1	3	1	1	2	2	1	1	3	2	2	3	1	3	1	1	2	2	1	1	3	1	471
2	1	3	1	3	1	1	1	2	2	2	3	1	2	1	1	1	3	1	1	1	3	1	3	472
1	1	1	3	2	2	2	1	2	3	1	1	3	2	2	1	2	2	3	1	3	2	1	3	473
1	1	1	2	1	3	2	3	2	1	1	3	2	1	1	1	3	1	3	1	2	3	1	2	474
2	1	2	3	1	2	3	1	2	2	2	1	3	2	2	1	2	1	1	3	1	3	2	3	475
2	1	3	1	2	1	1	1	2	3	2	2	1	2	3	1	2	3	1	3	2	1	1	3	476
1	3	1	2	2	3	1	2	2	3	2	3	1	2	3	1	2	2	2	3	2	1	2	1	477
2	2	1	1	3	1	1	3	1	1	2	2	3	2	1	2	1	2	3	1	3	1	3	2	478
3	2	1	3	1	1	2	3	2	2	2	1	3	1	3	2	2	3	1	1	2	1	2	1	479
3	1	3	1	1	1	2	1	3	2	1	1	3	1	1	3	2	1	1	1	2	1	3	1	480
1	2	2	3	1	1	3	2	2	3	2	2	1	2	3	2	3	1	1	3	1	2	2	2	481
2	1	1	1	2	3	2	2	3	2	3	2	1	3	1	3	2	1	1	2	2	1	3	1	482
1	1	1	2	1	1	3	1	3	2	2	2	3	1	3	1	1	3	2	2	3	2	2	2	483
1	3	2	2	3	2	1	1	2	1	1	3	1	1	3	2	3	1	2	2	2	1	1	3	484
3	2	2	1	3	1	1	2	3	2	1	2	1	2	1	3	1	3	2	2	1	3	1	2	485
2	2	3	1	2	1	2	2	3	1	1	1	3	1	3	1	1	1	3	2	2	1	2	3	486
2	2	1	1	1	3	1	3	1	3	1	1	1	2	3	2	2	2	3	1	2	2	1	3	487
2	3	2	3	1	1	2	2	2	3	1	3	2	1	2	2	1	3	2	1	1	3	1	1	488
2	1	1	2	2	2	3	1	1	2	3	2	3	1	1	1	3	2	2	3	2	2	1	3	489
1	2	3	2	3	2	2	2	3	1	1	1	3	1	2	3	1	2	3	1	2	2	2	1	490
1	1	3	2	2	1	2	3	2	2	3	1	2	1	2	2	3	1	3	2	3	1	1	1	491
2	1	3	1	2	1	1	1	3	1	1	3	1	2	1	3	1	3	1	2	2	2	1	3	492
3	1	2	3	1	1	2	3	2	1	3	1	2	1	2	1	2	3	2	1	1	2	3	1	493
3	1	1	3	1	1	2	1	3	2	2	2	1	2	3	2	1	1	1	2	3	1	2	3	494
3	2	1	3	2	1	2	1	2	1	3	2	2	1	1	1	3	1	2	3	1	3	2	2	495
3	2	2	1	2	2	2	3	2	3	2	1	2	3	1	2	2	1	2	3	1	2	2	3	496
1	3	1	3	1	2	2	1	3	1	1	1	2	2	3	1	3	1	3	1	1	2	2	1	497
3	2	1	2	3	1	2	1	3	1	3	2	2	2	1	2	1	3	2	3	1	2	1	1	498
3	2	2	1	3	1	1	1	3	1	1	2	3	1	1	1	2	2	3	1	1	3	2	1	499
1	1	3	1	1	2	3	1	3	1	1	2	1	2	1	3	1	3	1	2	3	1	1	2	500
1	1	1	3	1	3	1	1	2	1	3	2	3	2	2	2	1	1	3	1	1	3	1	2	501
3	1	2	3	2	3	2	2	1	2	2	3	1	2	1	3	1	1	1	2	2	1	3	1	502
2	1	3	1	3	2	2	1	2	1	3	1	3	1	2	1	2	2	3	2	1	2	3	1	503
3	1	3	1	3	2	2	3	1	1	2	1	1	3	2	2	1	1	1	3	1	2	1	2	504
1	3	1	2	1	2	3	1	1	1	2	1	3	1	2	2	3	2	2	1	3	1	3	1	505
3	1	3	2	3	1	1	2	1	3	1	1	1	3	1	2	1	2	3	2	2	1	1	2	506
1	1	1	3	1	3	1	2	1	2	2	3	1	1	3	1	3	1	1	2	1	1	1	3	507
3	2	2	1	2	1	3	1	1	2	1	1	3	2	2	3	2	1	1	1	3	2	3	2	508
2	3	1	2	1	3	2	1	2	3	1	2	1	1	2	3	2	3	2	2	2	1	2	3	509
2	2	2	3	2	2	3	2	2	1	1	3	2	1	2	3	2	3	1	2	2	2	1	3	510
2	1	1	1	3	2	3	2	2	3	2	3	2	2	1	1	1	3	1	2	2	1	1	3	511
2	3	2	3	2	2	2	3	1	2	2	3	1	2	2	1	1	2	3	2	2	1	2	3	512
1	2	2	1	1	2	3	1	1	2	3	1	3	2	3	2	2	3	2	1	1	2	3	2	513
2	1	3	1	2	3	2	2	2	3	2	3	1	3	2	2	2	3	1	2	1	2	2	1	514
3	1	1	2	3	1	1	2	1	3	2	1	1	2	1	3	1	2	3	1	2	2	2	3	515
1	1	2	1	3	2	3	2	3	2	2	3	2	2	1	2	1	2	3	1	2	2	1	3	516
2	1	3	1	2	2	1	3	1	1	3	1	2	3	2	2	3	2	3	2	1	2	2	1	517
1	1	2	3	2	3	2	3	2	3	2	2	1	1	1	2	3	1	1	2	2	2	3	2	518
3	1	1	2	2	1	1	3	2	1	2	1	2	3	1	3	2	3	2	1	3	1	1	1	519
2	2	1	2	2	3	2	3	2	3	2	1	1	3	2	1	3	2	3	2	1	1	1	2	520
3	2	1	3	2	1	1	1	3	1	3	1	1	2	2	3	2	2	2	1	3	2	1	2	521
1	1	3	2	2	2	3	2	1	1	3	1	1	3	2	1	3	2	2	3	1	1	2	1	522
1	3	2	2	1	2	1	3	2	1	2	1	3	2	1	3	2	1	2	1	3	1	3	1	523
3	1	1	1	3	1	1	1	2	3	2	3	2	1	2	1	3	2	2	2	1	1	2	3	524
2	2	3	2	3	1	3	2	1	1	2	3	1	1	2	3	1	2	3	2	1	2	2	1	525
3	2	1	3	1	3	2	2	3	2	1	1	1	2	1	3	1	3	1	1	2	1	1	1	526
1	2	2	1	1	2	3	2	1	3	1	2	2	3	2	1	1	3	1	3	1	2	1	3	527
2	2	1	3	2	3	2	3	2	2	2	3	2	1	3	1	2	1	3	1	1	2	2	1	528
1	3	1	3	1	3	2	2	2	3	2	3	2	1	2	1	2	3	2	1	2	1	1	1	529
2	2	1	1	3	2	2	2	1	3	2	3	1	3	1	2	2	2	3	2	2	1	1	3	530
1	2	3	1	1	3	2	2	2	1	2	2	3	1	1	2	1	3	2	1	3	2	3	1	531
1	2	1	2	2	2	3	2	3	2	2	3	2	1	2	3	2	2	2	3	2	3	1	1	532
1	1	1	3	2	3	2	2	2	1	2	1	3	1	1	3	1	2	2	2	3	1	2	3	533
1	1	3	1	3	1	2	1	2	3	1	2	2	2	3	2	2	1	3	2	2	3	2	1	534
1	1	3	1	1	3	1	1	1	2	3	1	3	2	3	1	2	1	1	2	3	2	1	1	535
2	1	3	2	3	2	2	2	3	1	2	1	2	3	2	2	1	1	3	1	1	3	2	2	536
3	2	1	3	1	1	1	3	2	3	1	2	1	3	1	2	2	1	3	2	1	1	2	1	537
3	1	2	1	1	1	2	3	2	2	1	1	3	2	2	1	3	2	1	2	3	1	2	3	538
1	3	1	2	2	1	3	1	1	3	1	1	2	2	3	2	2	2	1	3	1	1	2	3	539
1	2	1	2	2	2	3	1	3	1	1	3	2	3	2	3	1	1	1	2	3	1	1	2	540
2	3	1	3	2	1	1	1	2	1	3	2	2	2	1	2	3	1	3	2	1	3	2	1	541
2	2	1	3	1	3	1	3	2	1	3	1	2	1	1	1	3	1	2	2	2	3	1	2	542
1	2	2	3	2	2	2	1	1	3	2	2	3	2	2	3	1	2	1	1	3	1	2	3	543
3	2	2	3	2	1	1	1	3	2	2	1	1	1	3	2	3	2	3	1	1	2	2	2	544
1	2	1	3	1	2	2	3	2	3	2	3	2	2	2	3	2	2	1	2	1	3	2	1	545
3	2	1	1	3	2	2	1	2	2	3	1	3	1	1	2	3	1	2	1	1	2	1	3	546
2	1	3	1	2	2	1	3	2	2	3	1	2	1	1	3	2	3	2	3	2	1	1	2	547
1	1	1	2	3	2	1	1	1	2	3	1	1	3	1	3	2	3	2	2	2	3	2	2	548
3	1	2	1	3	1	1	3	1	1	1	2	3	2	1	2	1	2	1	3	2	3	1	2	549
2	1	2	1	3	1	3	2	3	2	1	2	3	2	2	1	2	3	1	2	1	1	1	3	550
2	1	2	3	1	1	3	2	3	1	2	1	1	3	1	2	3	1	1	3	1	1	2	2	551
2	3	2	2	3	1	3	1	1	2	1	3	2	1	1	3	1	3	1	1	2	2	2	1	552
2	1	3	1	2	1	1	2	3	2	3	1	1	3	2	1	1	2	1	1	3	2	3	1	553
3	2	1	2	2	1	2	3	1	2	3	1	2	1	3	2	1	3	2	1	1	3	2	1	554
1	3	1	2	1	2	3	1	2	2	2	1	3	2	1	2	2	3	1	1	2	3	2	3	555
1	1	2	2	1	1	3	2	2	2	3	2	1	3	1	3	2	3	1	2	2	2	3	1	556
1	1	3	1	1	1	2	1	3	1	2	3	2	1	3	2	1	1	3	1	2	3	2	2	557
2	2	3	1	3	1	1	3	2	2	3	2	2	3	2	1	1	2	1	1	3	1	1	2	558
1	3	2	3	2	3	1	1	1	2	1	3	2	3	1	1	1	3	2	2	2	1	1	1	559
2	2	2	1	2	3	2	1	3	2	1	3	1	2	2	2	1	2	3	2	3	1	1	3	560
1	2	2	1	1	2	3	1	3	1	1	1	2	2	1	3	2	3	2	3	2	2	1	3	561
1	2	3	2	2	1	1	2	1	3	2	3	1	2	1	3	2	1	1	1	3	2	3	1	562
2	1	2	3	2	2	3	1	2	1	1	1	2	3	1	2	2	1	2	3	1	3	2	3	563
2	2	1	2	2	1	3	1	3	2	2	3	2	3	2	3	2	3	1	2	1	2	1	2	564
2	3	2	2	3	2	2	1	2	3	1	2	2	3	1	3	2	2	1	3	1	1	2	1	565
1	1	2	2	2	3	1	3	2	2	1	1	3	1	1	3	1	1	3	2	3	2	1	1	566
1	1	1	3	1	2	1	1	1	3	2	2	1	1	3	2	3	2	2	2	3	2	1	3	567
2	3	2	2	3	1	3	1	2	3	1	2	1	2	2	3	2	1	2	1	1	3	2	2	568
2	1	1	1	2	1	3	2	3	1	1	2	3	1	3	2	2	1	2	1	3	1	3	2	569
1	2	1	3	1	2	3	2	2	1	2	3	1	2	1	3	2	2	1	3	2	2	1	3	570
3	2	2	1	1	3	2	3	1	1	3	1	2	1	2	3	2	1	2	2	3	2	2	1	571
2	1	1	3	1	1	1	3	2	1	1	1	3	2	2	2	3	2	1	3	1	2	3	2	572
1	1	3	1	3	1	1	1	3	2	2	2	3	1	2	2	3	1	1	2	1	1	1	3	573
1	2	1	2	2	1	3	1	2	3	2	3	1	3	2	2	1	2	1	2	3	2	3	2	574
1	3	2	2	2	3	1	3	2	2	2	1	3	2	1	2	2	3	2	3	1	1	2	1	575
1	2	3	2	2	1	1	1	2	3	1	3	1	3	1	2	2	3	2	3	2	1	2	1	576
2	1	1	1	2	3	2	2	3	2	3	1	2	2	1	2	2	3	2	3	1	3	1	2	577
2	1	1	3	1	1	2	2	3	1	1	3	2	1	1	3	1	3	2	2	1	2	2	3	578
1	3	1	3	1	2	1	3	1	1	2	2	1	1	3	2	2	2	3	2	2	3	1	2	579
3	1	1	3	1	1	2	3	2	2	1	1	3	1	1	1	2	1	2	3	2	1	1	3	580
2	1	2	2	2	3	2	3	1	2	2	1	1	3	1	1	3	2	2	3	1	3	1	1	581
1	3	2	2	1	3	1	1	2	2	2	3	2	3	2	1	3	2	1	3	1	1	2	2	582
1	1	3	2	2	2	1	2	2	3	2	2	3	1	2	3	2	2	3	2	1	2	2	3	583
3	1	1	2	3	1	3	2	2	2	1	1	3	1	3	2	2	2	1	2	1	3	2	1	584
1	3	2	3	1	1	3	1	2	2	3	2	1	2	3	2	1	3	2	1	2	1	1	1	585
1	3	2	2	3	1	1	1	2	3	1	3	2	1	2	2	1	1	3	2	1	1	2	3	586
1	2	3	2	3	2	2	1	2	2	2	3	1	3	1	2	3	1	3	2	1	1	2	2	587
1	1	1	2	1	3	2	3	2	2	3	2	2	3	1	1	3	2	2	3	2	2	1	2	588
3	2	1	3	1	3	1	1	1	3	1	2	1	2	1	2	3	2	1	3	2	2	2	1	589
3	1	3	1	3	2	1	2	2	2	3	1	2	3	1	1	2	3	1	2	2	1	2	1	590
3	1	3	2	1	2	1	1	3	2	2	2	1	3	2	3	2	1	2	1	2	2	3	1	591
1	2	1	1	2	3	2	3	1	2	2	1	2	2	3	1	2	2	3	1	3	1	3	1	592
2	2	1	3	2	2	3	2	2	1	2	3	2	3	1	3	1	3	2	1	1	2	1	1	593
1	1	1	2	3	1	3	2	1	2	1	2	2	3	1	1	2	2	3	2	3	1	2	3	594
1	1	2	2	1	3	1	1	3	2	1	1	3	2	1	3	1	3	2	2	2	1	1	3	595
2	3	2	1	1	3	2	2	2	1	1	1	3	2	1	1	3	1	1	1	2	3	2	3	596
3	1	1	1	2	3	1	2	1	1	3	2	2	3	1	2	1	2	1	1	3	1	1	3	597
1	1	2	3	1	3	2	1	3	2	2	2	3	2	1	2	2	2	3	1	3	2	2	2	598
1	3	2	3	1	1	2	3	2	1	1	3	1	2	2	1	2	3	2	1	2	2	2	3	599
3	2	1	1	2	2	3	1	1	2	2	3	1	1	1	3	1	2	1	1	3	2	3	2	600
2	1	2	3	2	2	2	1	1	3	2	1	3	2	3	1	1	1	2	1	3	1	3	2	601
3	2	1	2	2	3	1	1	1	2	2	3	1	1	2	2	1	3	1	1	3	2	1	3	602
1	1	2	1	2	3	2	1	1	2	3	2	1	3	2	2	3	1	1	1	3	2	3	1	603
2	3	1	1	2	1	2	2	3	1	3	1	1	2	2	1	2	3	1	3	1	3	2	2	604
2	1	3	2	3	2	1	1	1	2	3	1	2	3	1	1	3	1	1	1	3	2	1	2	605
3	2	1	2	3	2	3	2	1	1	1	3	1	1	1	2	2	2	3	1	2	3	2	1	606
1	1	2	2	3	2	2	2	3	1	1	1	3	2	2	2	3	2	2	3	1	3	1	1	607
1	1	2	2	3	2	2	2	3	1	3	2	1	3	2	1	2	2	1	3	2	1	3	2	608
2	1	1	2	2	3	1	3	2	2	2	3	1	1	2	1	1	3	1	3	1	3	2	2	609
2	3	2	2	3	1	2	2	3	2	1	1	3	2	3	2	2	2	1	2	2	3	2	2	610
3	1	1	1	2	2	2	3	2	3	1	3	2	1	2	3	2	1	2	2	2	3	1	1	611
2	1	1	3	1	1	2	3	1	1	2	3	2	3	1	1	3	2	3	1	1	2	1	2	612
2	1	1	2	3	2	3	1	1	3	2	2	2	3	2	3	1	1	1	3	1	2	1	2	613
2	2	3	2	1	2	1	2	3	1	1	1	3	2	1	1	3	1	1	3	1	1	3	2	614
2	1	3	1	3	1	3	1	1	3	1	1	3	1	1	1	2	1	1	3	1	1	2	1	615
1	2	2	2	3	1	1	1	2	3	2	2	1	1	2	3	1	3	1	3	1	3	1	2	616
2	2	3	2	3	2	3	2	1	2	1	2	1	3	2	1	2	2	1	3	1	1	2	3	617
1	2	2	3	2	2	1	3	2	1	2	2	3	1	2	3	2	3	1	1	3	2	2	1	618
2	3	2	2	2	3	2	1	2	2	2	3	1	1	2	3	1	1	1	2	3	1	1	3	619
2	3	2	2	1	3	1	2	2	3	2	3	2	2	1	1	1	2	3	2	1	3	2	2	620
2	1	2	1	3	1	3	2	1	2	2	3	2	1	2	1	3	1	3	1	3	1	1	1	621
1	1	1	2	1	3	2	1	1	3	1	1	2	3	2	1	3	2	2	3	2	2	3	1	622
2	3	1	3	2	3	2	3	1	2	2	2	1	2	3	1	2	2	1	1	3	2	2	1	623
1	3	1	1	2	2	2	3	2	2	3	2	1	3	2	3	2	2	1	2	3	1	2	2	624
3	1	2	2	3	1	1	3	1	1	1	3	1	1	1	2	1	3	2	2	2	3	1	1	625
3	1	2	1	1	2	1	3	1	3	1	1	2	1	3	2	1	3	1	3	2	2	1	1	626
3	1	2	2	3	1	1	1	2	2	2	3	2	1	3	2	2	1	2	1	3	2	3	1	627
3	1	2	2	2	1	1	3	1	1	3	1	2	3	1	1	2	1	1	2	3	2	1	3	628
2	2	2	3	1	3	1	3	1	1	1	3	2	1	3	1	1	2	1	1	3	1	2	1	629
3	1	2	2	1	1	3	1	3	2	1	1	1	2	3	1	3	2	1	2	1	1	3	1	630
2	2	2	3	1	2	1	3	1	1	2	2	3	1	1	1	2	2	2	3	1	3	1	3	631
2	3	1	1	3	1	1	3	1	3	2	3	2	2	1	2	1	1	3	1	2	2	2	1	632
3	2	3	1	1	1	2	3	1	2	2	2	1	3	1	3	2	1	1	2	1	1	3	2	633
1	1	1	2	1	1	3	1	1	2	1	3	1	3	1	3	1	3	1	1	1	3	2	2	634
3	2	2	3	2	1	1	1	3	2	1	1	2	1	3	1	3	1	1	1	2	2	1	3	635
1	3	2	3	1	2	2	2	1	3	1	2	2	1	2	3	2	3	1	2	3	1	2	2	636
1	3	1	3	2	1	2	1	3	2	2	2	1	3	1	2	2	2	1	2	3	2	1	3	637
1	2	3	1	2	2	1	3	1	2	1	3	2	3	1	1	1	2	2	3	2	2	1	3	638
1	2	3	1	1	1	2	3	2	1	2	2	1	3	2	2	2	1	3	1	3	2	2	3	639
1	2	1	2	2	3	1	3	2	3	1	3	1	3	2	2	1	2	2	3	2	2	1	1	640
1	3	1	2	3	2	3	2	1	2	2	3	1	1	2	2	1	1	3	1	1	3	2	2	641
2	1	1	2	3	2	3	2	2	3	1	2	1	3	1	1	2	1	3	1	3	1	2	1	642
1	1	1	2	2	1	3	2	2	3	1	1	1	3	2	1	2	3	1	3	1	1	1	3	643
2	2	2	1	3	1	3	2	2	3	1	1	3	1	1	1	2	3	2	2	1	1	2	3	644
3	1	2	2	3	2	2	3	1	2	2	1	2	2	3	1	2	3	1	1	2	2	2	3	645
2	3	2	2	3	2	2	3	2	2	3	1	1	2	2	3	1	1	3	1	1	2	2	1	646
1	2	2	1	1	3	2	1	1	3	1	1	2	2	3	1	3	1	3	2	2	2	3	1	647
3	2	1	2	3	2	2	3	2	1	1	2	3	2	1	2	2	1	1	3	1	1	1	3	648
2	1	3	2	2	3	2	3	1	2	2	2	1	2	3	2	1	1	2	3	1	2	2	3	649
2	3	1	2	1	1	2	3	1	1	1	3	2	2	2	1	2	1	3	1	3	1	3	1	650
3	2	1	1	3	1	2	2	3	2	2	2	3	2	1	2	3	1	2	1	1	3	1	2	651
2	2	3	1	1	2	2	1	1	3	1	3	2	1	1	3	1	2	3	2	2	2	1	3	652
1	1	3	2	3	2	2	2	3	2	2	2	1	3	1	3	2	1	1	1	3	1	2	1	653
1	3	1	3	1	3	1	2	1	1	1	3	2	1	2	1	3	1	1	3	2	2	1	1	654
1	2	2	1	2	3	1	1	2	1	3	2	2	1	3	1	1	1	3	1	3	1	3	2	655
2	2	3	2	2	3	1	2	1	2	2	1	3	1	3	1	1	2	3	2	3	2	2	2	656
2	2	2	1	2	2	3	1	3	1	3	2	2	2	3	2	2	1	2	2	2	3	2	3	657
1	3	2	3	2	2	1	1	3	1	1	3	2	2	3	1	2	2	1	2	2	3	1	2	658
3	1	3	1	1	1	2	3	1	2	2	3	1	1	2	3	2	2	3	1	2	1	1	2	659
3	1	2	1	1	3	2	1	2	2	1	3	2	1	2	3	1	3	2	3	2	1	1	2	660
2	2	2	3	1	2	2	2	1	1	3	1	3	2	3	2	2	3	1	1	2	3	2	1	661
1	1	3	2	2	1	3	2	1	1	1	2	1	3	1	3	2	1	3	1	1	1	3	1	662
3	2	1	1	1	3	2	1	2	3	1	1	2	1	2	3	2	3	1	1	1	2	3	2	663
2	1	1	2	1	1	3	2	3	2	3	2	2	3	2	3	1	1	2	3	2	1	1	2	664
1	1	1	3	1	2	2	2	1	3	1	3	2	1	3	1	1	1	3	1	3	1	1	2	665
2	2	1	3	2	2	2	3	1	3	2	2	3	1	1	1	2	1	3	2	1	1	1	3	666
2	1	1	2	1	3	2	1	2	3	1	3	2	1	1	3	1	2	2	3	1	1	1	3	667
3	1	1	3	1	2	2	1	3	1	2	2	3	1	2	3	2	2	1	3	2	2	1	1	668
2	1	1	1	3	1	3	1	3	1	1	3	2	2	1	3	2	1	1	2	1	3	1	1	669
2	1	1	3	2	1	2	3	1	3	1	1	1	2	3	1	2	3	2	3	2	2	1	2	670
3	1	3	2	2	2	3	2	2	2	3	2	2	1	3	2	2	1	2	2	3	1	2	1	671
1	1	3	2	1	1	1	3	1	1	1	2	3	2	2	1	1	3	1	3	2	1	3	2	672
1	2	3	1	3	1	1	2	2	3	2	2	3	2	2	3	1	1	1	2	3	2	1	1	673
2	2	1	3	1	2	2	1	3	1	3	2	1	3	2	1	3	1	1	3	1	1	2	1	674
2	1	3	2	3	1	2	3	1	1	3	1	1	3	2	2	1	3	1	1	1	2	2	1	675
2	1	1	2	3	2	1	3	2	1	1	2	3	2	3	1	2	3	1	2	1	1	3	2	676
2	2	3	1	3	1	1	1	3	1	1	2	1	1	3	2	1	3	2	3	2	1	1	1	677
2	1	1	2	3	1	3	2	3	1	3	1	2	2	1	2	1	3	1	2	2	3	2	2	678
3	2	1	2	1	1	3	1	1	1	2	3	2	3	2	3	2	2	2	3	1	2	2	1	679
3	2	3	1	1	2	3	2	3	2	2	1	1	2	3	1	1	3	1	2	1	2	1	2	680
3	1	1	1	3	2	2	1	2	2	1	3	2	1	3	2	2	1	1	1	3	1	2	3	681
2	1	3	1	1	2	2	3	2	3	2	2	2	3	1	2	1	1	3	2	3	1	2	1	682
2	3	1	2	2	2	1	3	1	2	2	3	1	3	1	3	2	2	1	1	1	2	3	1	683
1	2	2	1	2	2	3	1	3	2	2	2	3	1	1	2	3	2	2	3	1	2	1	3	684
1	2	1	3	2	1	3	2	2	1	2	3	2	2	2	3	1	2	2	2	1	3	2	3	685
1	2	1	3	1	1	3	1	1	3	1	1	2	1	1	1	3	2	2	1	3	1	3	1	686
3	1	2	3	2	2	3	1	1	1	3	2	1	1	2	3	1	1	2	2	2	3	2	1	687
3	1	3	1	2	2	3	1	2	1	3	2	1	3	1	1	1	2	3	1	2	1	1	1	688
2	3	1	3	1	3	1	1	2	1	1	1	3	2	1	2	3	1	1	2	2	2	3	1	689
2	1	2	1	1	1	3	1	2	3	1	2	3	2	3	1	1	2	2	1	3	2	1	3	690
2	2	1	2	3	2	1	1	3	1	1	2	3	2	2	2	3	1	3	1	3	1	1	1	691
1	3	2	1	1	1	2	3	1	2	3	1	1	2	3	1	2	1	2	3	1	2	3	1	692
3	1	1	1	2	2	2	3	2	3	2	2	1	1	1	3	2	2	3	1	1	2	3	1	693
3	1	2	3	1	1	2	3	1	2	2	3	2	3	2	2	2	1	1	3	2	1	2	1	694
3	1	1	1	2	1	1	3	2	3	1	3	1	3	2	2	1	1	2	3	1	1	1	2	695
2	3	2	2	3	1	1	1	2	1	3	2	2	1	2	2	1	3	2	2	2	3	2	3	696
2	2	2	3	1	3	1	3	2	1	2	1	2	2	3	1	2	1	2	3	1	3	1	1	697
1	2	2	3	2	3	2	3	2	1	1	1	3	2	1	1	3	1	2	2	2	1	1	3	698
2	1	2	1	3	2	2	2	3	1	1	3	2	3	2	3	1	2	3	2	1	2	2	2	699
3	2	3	1	1	3	2	2	1	2	1	3	2	3	2	1	2	1	1	1	3	1	1	2	700
3	2	1	2	3	2	2	3	1	1	2	1	3	2	1	1	1	2	1	3	1	2	2	3	701
2	2	1	3	1	1	1	3	2	3	2	3	1	2	2	2	3	2	3	2	1	2	2	2	702
2	2	2	1	3	2	1	1	2	1	2	3	2	1	1	3	1	3	1	2	3	2	3	1	703
1	3	2	1	2	3	2	1	2	1	3	1	2	3	1	2	3	2	2	2	3	2	2	2	704
1	2	2	2	1	1	3	2	1	1	1	3	2	3	2	1	3	1	3	1	2	1	1	3	705
1	2	2	2	3	2	3	2	2	3	1	1	2	2	3	2	1	1	1	3	2	3	1	1	706
1	2	3	2	2	1	2	2	1	3	1	2	2	3	2	3	1	2	3	1	1	2	3	1	707
2	1	3	2	1	3	2	1	3	1	1	2	1	2	3	1	1	1	2	2	1	3	1	3	708
2	2	2	1	1	2	3	1	3	1	1	3	1	3	2	2	1	3	1	3	2	1	2	1	709
1	1	1	3	2	2	2	1	3	2	1	3	1	3	2	3	2	1	2	3	2	1	1	1	710
1	2	1	2	1	2	3	1	2	1	3	2	1	3	1	3	2	1	3	1	2	2	1	3	711
2	3	1	3	1	1	3	2	2	1	1	2	2	3	2	1	2	1	3	1	2	2	3	1	712
2	1	2	1	3	1	3	1	2	3	2	2	1	2	1	2	3	1	1	3	2	2	3	2	713
1	1	1	2	2	2	3	2	2	1	1	3	2	2	3	2	2	3	2	2	3	2	2	3	714
2	2	3	2	2	3	1	1	3	1	2	3	1	1	1	3	2	1	3	1	1	2	2	1	715
1	1	3	1	3	1	2	1	1	3	2	1	3	2	3	2	2	2	1	2	3	2	2	2	716
1	1	2	1	1	3	1	1	3	1	1	3	2	3	1	1	1	3	1	2	2	3	1	2	717
2	1	1	3	2	2	1	1	1	3	2	2	3	1	2	3	1	2	2	3	1	2	1	3	718
1	2	1	2	1	1	3	1	2	1	1	3	1	3	2	3	2	1	1	3	2	3	1	2	719
3	2	2	1	1	1	2	3	2	2	3	2	2	3	2	2	2	1	1	3	2	3	1	2	720
3	1	3	2	2	1	1	3	2	2	1	2	2	1	3	2	2	1	1	3	1	1	3	2	721
2	1	2	2	1	3	1	3	2	2	2	3	1	3	1	1	2	1	1	3	2	1	3	2	722
2	1	1	2	3	2	2	3	2	2	1	2	3	2	3	2	2	1	3	1	2	3	2	2	723
3	1	1	1	3	2	2	3	1	2	1	3	1	1	2	3	2	1	1	2	3	2	2	2	724
2	3	1	2	1	3	1	2	3	1	1	2	2	3	1	2	2	3	1	2	2	1	3	2	725
1	2	3	1	2	1	3	1	3	2	1	1	1	3	1	1	2	1	1	3	2	2	3	2	726
1	3	2	1	1	3	2	3	2	2	1	3	1	2	1	3	2	1	2	2	3	1	1	2	727
1	2	3	2	1	3	1	2	2	1	1	1	3	2	1	3	2	3	2	1	2	3	2	2	728
2	2	1	2	2	3	1	2	1	1	2	3	1	3	1	3	1	3	2	2	1	1	1	3	729
1	2	2	2	3	2	2	1	2	3	1	2	1	1	1	2	3	2	3	2	1	3	2	3	730
2	2	3	1	1	3	1	1	1	2	1	1	3	1	3	2	1	1	2	1	1	3	1	3	731
2	3	2	3	2	1	1	2	1	1	3	2	1	3	2	1	1	3	1	2	2	1	3	1	732
1	2	3	1	1	1	3	2	2	1	3	1	3	2	2	2	1	2	3	1	2	1	1	3	733
1	2	2	1	3	2	2	1	1	3	1	3	1	3	2	2	2	3	2	1	3	1	2	2	734
2	3	2	1	3	2	1	2	2	3	2	1	2	3	1	2	2	1	1	1	3	2	3	2	735
1	3	2	2	3	1	2	1	1	1	3	1	1	3	1	1	3	1	3	2	1	2	1	2	736
3	2	1	1	2	3	1	3	1	2	1	1	1	3	1	3	1	3	1	2	1	1	2	2	737
2	3	2	3	2	2	3	1	1	3	1	2	1	1	1	3	2	2	2	1	2	3	1	2	738
1	1	3	1	1	3	1	3	2	1	3	2	2	1	3	1	1	2	2	3	1	2	2	1	739
3	1	1	2	3	1	1	3	1	2	3	1	1	3	2	2	2	3	2	2	1	1	2	1	740
1	1	1	2	2	3	2	2	3	1	3	1	2	1	1	3	1	2	1	3	2	3	1	2	741
2	3	1	2	2	3	2	2	2	1	1	2	3	1	2	3	2	3	2	3	1	2	2	1	742
1	2	3	1	1	3	2	1	2	2	3	2	2	3	1	3	2	3	1	2	2	2	1	1	743
3	2	3	2	1	1	1	2	3	2	2	2	3	1	3	1	2	3	2	1	2	1	2	2	744
1	1	2	2	3	1	2	3	1	3	2	2	2	1	1	1	3	1	3	2	2	3	1	2	745
2	2	2	3	2	3	2	1	1	2	1	2	3	1	2	2	3	1	3	1	3	2	2	2	746
3	2	1	3	2	1	3	1	2	3	1	2	2	1	1	3	1	1	3	1	2	1	1	1	747
2	2	2	1	1	2	3	2	3	1	1	1	2	2	2	3	2	2	3	2	3	1	3	2	748
3	2	1	1	1	3	1	1	2	2	1	3	1	2	1	1	1	3	1	3	2	3	1	2	749
1	1	2	1	3	2	2	1	1	3	2	2	2	1	1	3	1	3	2	2	3	2	3	2	750
3	2	3	2	3	1	2	3	2	2	2	1	2	1	3	1	2	2	2	3	2	2	1	2	751
3	2	1	2	1	3	2	3	2	3	1	2	2	1	3	1	2	2	2	3	2	1	1	1	752
3	2	2	3	2	1	1	3	1	1	1	3	1	2	1	2	3	2	1	1	3	1	1	1	753
1	2	1	2	2	1	3	1	2	2	3	2	1	1	1	3	1	3	1	3	2	3	1	1	754
3	1	3	2	3	1	2	1	2	2	3	1	1	1	2	2	1	3	1	2	2	3	2	1	755
2	1	1	3	1	1	3	2	2	1	1	1	3	1	1	3	1	3	1	3	2	2	2	1	756
3	1	2	3	2	2	1	3	1	2	1	1	1	3	2	2	2	1	1	3	2	1	3	2	757
3	2	3	1	2	2	3	2	1	2	3	1	3	1	1	1	2	3	2	2	1	1	1	2	758
2	3	1	2	2	1	2	2	3	2	1	1	3	1	1	1	3	1	2	2	3	1	3	1	759
1	1	3	1	1	2	2	3	2	3	2	1	1	3	2	2	2	1	2	3	1	3	2	1	760
2	2	3	2	1	2	2	2	1	3	1	1	3	1	2	2	2	3	2	1	3	1	2	3	761
2	1	2	1	2	3	2	2	2	3	2	3	2	1	1	3	1	1	3	1	1	1	2	3	762
3	1	2	1	1	2	3	2	3	2	3	1	1	2	2	2	3	2	3	1	1	2	1	1	763
2	2	1	3	1	1	1	2	3	2	3	1	3	1	2	2	2	1	1	3	1	3	2	2	764
1	3	2	3	2	1	3	1	1	2	2	2	3	2	1	2	2	2	1	3	2	2	3	2	765
2	1	3	2	2	1	1	3	1	2	1	3	1	3	2	1	1	1	2	3	1	2	1	3	766
3	1	1	3	2	3	1	2	1	2	2	3	2	1	1	1	2	2	3	1	2	1	1	3	767
3	2	1	1	2	2	3	2	3	2	2	1	3	1	2	2	2	1	1	3	1	1	3	2	768
2	3	1	2	1	2	2	2	3	2	3	1	1	2	2	3	1	2	1	3	2	1	2	3	769
1	1	3	2	1	1	1	3	1	3	1	2	1	2	1	3	2	2	1	1	3	2	2	3	770
1	2	2	1	3	2	2	1	1	3	2	2	1	2	2	2	3	2	3	1	3	2	3	1	771
1	3	1	2	3	1	1	3	2	1	3	2	2	2	1	2	3	1	1	2	2	1	3	1	772
2	3	1	3	2	2	1	3	2	2	1	1	3	2	3	1	2	1	3	2	2	1	1	1	773
2	2	1	2	2	3	2	1	3	1	2	2	2	1	3	1	3	1	1	3	1	2	3	1	774
2	1	2	2	2	3	2	3	2	2	2	3	2	2	3	1	2	2	1	3	1	2	1	3	775
3	2	1	2	1	1	2	3	2	3	2	3	2	3	1	1	1	3	2	2	1	2	1	1	776
2	1	2	1	2	3	2	2	3	1	3	2	1	2	1	1	1	3	1	3	1	3	1	1	777
2	2	1	3	2	2	1	3	2	2	2	1	1	1	3	1	2	2	3	2	3	1	3	2	778
2	2	2	1	3	1	1	2	1	1	3	2	3	1	2	3	2	3	1	2	3	1	1	1	779
1	3	1	3	2	1	1	2	3	2	3	2	1	1	1	2	1	3	2	2	1	3	1	2	780
2	3	2	3	1	2	1	1	1	3	1	3	1	1	1	2	2	1	3	2	2	3	2	2	781
3	1	1	2	2	2	1	3	2	3	1	1	2	3	2	2	2	3	1	3	1	2	2	1	782
2	3	2	3	1	2	3	2	3	2	1	1	3	2	1	2	1	2	3	1	1	1	2	2	783
2	2	3	2	3	1	1	2	3	1	2	2	1	1	2	3	1	1	2	1	3	1	1	3	784
1	1	2	3	2	2	3	2	2	2	1	3	1	2	2	3	1	3	1	1	1	3	2	2	785
1	3	1	2	2	3	2	3	2	2	1	3	2	1	2	2	1	3	2	1	2	1	1	3	786
2	2	3	1	2	3	2	1	2	2	1	3	1	1	1	3	2	2	2	1	2	3	2	3	787
2	1	2	3	1	2	2	3	2	3	2	3	2	2	1	3	1	3	1	1	2	2	2	1	788
2	1	3	2	3	2	1	3	1	2	1	2	2	2	3	1	2	1	3	2	2	1	2	3	789
1	3	2	2	2	1	1	2	3	2	3	2	2	2	1	3	2	2	3	2	2	1	2	3	790
2	3	2	3	2	1	1	1	3	2	1	3	1	1	1	3	2	1	1	1	3	1	2	2	791
3	2	2	1	2	3	1	2	1	2	1	3	2	3	1	3	2	2	3	2	2	1	2	2	792
2	2	2	3	1	2	2	3	1	1	2	3	2	2	1	1	2	1	3	2	3	2	3	2	793
1	3	1	3	2	1	2	2	1	3	2	1	3	2	2	1	2	2	3	2	1	1	3	2	794
2	1	1	3	2	1	3	1	1	1	3	1	1	3	1	1	3	1	2	1	2	2	2	3	795
1	3	1	1	1	3	1	3	1	1	2	2	1	2	3	2	1	1	2	3	1	1	1	3	796
2	2	1	3	1	2	2	2	3	2	2	1	3	2	3	2	3	1	2	2	2	1	1	3	797
3	1	2	3	1	2	2	1	1	3	1	2	1	2	1	3	1	3	1	2	1	3	2	2	798
1	2	1	2	2	2	3	1	3	2	3	1	2	2	1	1	3	1	3	2	1	1	2	3	799
2	3	2	1	2	2	3	2	3	1	3	2	2	1	1	3	2	1	2	1	1	3	2	2	800
1	1	2	2	2	1	3	2	1	3	1	1	1	3	2	3	2	2	3	2	3	2	2	2	801
3	2	2	1	3	1	1	3	1	2	2	1	1	3	2	2	3	1	1	2	1	1	2	3	802
2	1	1	1	3	2	1	2	3	2	3	1	3	1	2	3	1	2	2	2	1	2	3	2	803
2	3	1	1	1	2	3	1	2	2	1	1	1	3	1	2	3	1	1	3	1	2	3	1	804
2	2	1	2	2	1	3	1	2	3	2	2	3	1	3	2	3	2	2	2	3	2	2	2	805
2	1	3	2	3	2	2	2	1	1	1	3	1	3	2	1	3	2	1	2	1	2	3	1	806
1	3	2	2	1	2	1	1	3	2	1	1	1	2	3	1	2	3	2	2	3	1	2	3	807
2	2	1	1	3	1	3	1	3	1	1	1	2	1	1	3	2	3	2	1	2	2	3	2	808
3	1	2	1	2	2	3	1	1	1	2	3	2	3	2	1	1	1	2	3	1	2	3	1	809
1	2	3	1	2	3	1	1	2	2	1	1	3	1	1	1	3	1	1	1	3	1	3	1	810
3	1	1	2	1	3	2	2	2	3	1	2	2	2	3	2	3	2	2	2	3	2	2	1	811
1	3	2	2	3	2	2	2	1	3	1	2	2	2	3	1	2	2	2	3	2	1	1	3	812
3	2	1	2	3	1	3	1	2	2	2	3	1	2	1	2	1	1	3	1	2	2	1	3	813
2	2	2	1	2	1	3	2	3	1	3	2	1	2	1	3	2	3	1	2	3	1	2	2	814
2	1	2	1	2	3	2	3	1	1	3	1	2	1	2	1	1	3	2	3	2	2	2	3	815
1	2	2	3	1	2	1	3	1	2	3	1	2	1	3	2	1	1	2	2	3	1	3	2	816
2	3	1	2	1	3	1	2	3	2	3	1	1	3	1	1	2	2	2	3	1	2	2	2	817
3	1	1	3	1	2	1	2	2	3	1	1	1	3	1	1	2	2	2	3	1	2	3	2	818
3	1	2	3	2	2	2	1	3	2	3	2	1	3	1	2	1	2	1	3	1	2	2	2	819
3	1	1	2	1	2	2	3	1	3	2	2	1	2	1	1	3	2	1	3	2	1	3	2	820
1	3	2	3	1	3	2	1	1	3	2	1	1	2	1	3	1	1	1	3	1	2	2	2	821
3	2	1	3	1	1	2	1	1	3	2	1	1	2	2	2	3	2	3	1	3	1	2	1	822
3	1	3	2	2	1	2	2	2	3	1	3	1	2	2	2	1	3	1	2	3	2	2	2	823
3	1	1	1	2	3	1	2	3	1	2	2	3	1	1	2	2	2	1	3	1	3	1	2	824
1	1	1	2	1	3	2	3	2	3	1	3	1	1	2	1	3	2	2	1	1	3	2	1	825
1	2	3	2	3	2	2	1	1	3	2	2	3	2	1	3	1	1	3	1	1	2	1	1	826
1	2	1	1	2	3	1	3	2	2	1	1	2	1	3	2	3	2	1	1	3	1	1	3	827
1	2	1	1	3	1	3	1	2	3	2	2	2	1	1	3	2	2	1	3	1	1	1	3	828
2	3	2	2	1	3	2	3	2	2	1	3	1	1	1	2	1	2	3	1	1	1	3	1	829
2	2	2	1	3	1	1	3	1	2	2	3	2	2	1	3	1	2	1	1	3	2	2	3	830
3	2	3	2	1	1	2	3	2	1	2	1	1	3	1	2	1	3	2	2	1	1	3	2	831
2	1	2	2	1	3	1	3	1	3	1	1	1	2	2	3	2	1	3	1	3	1	2	2	832
2	1	3	2	3	1	3	1	2	1	1	1	3	2	1	1	1	3	2	2	2	1	2	3	833
2	2	3	2	3	1	1	1	3	2	2	1	1	3	2	1	1	3	2	2	1	3	2	2	834
1	1	1	3	2	3	2	1	1	3	2	2	3	1	1	3	1	1	2	1	2	2	3	1	835
3	1	1	2	1	3	1	3	2	3	2	2	1	2	2	2	3	1	1	1	2	1	3	1	836
2	1	2	1	1	3	1	3	1	3	1	3	1	2	1	1	3	2	1	1	2	1	1	3	837
2	3	1	3	2	3	1	1	1	2	2	3	1	2	1	3	1	3	2	1	1	1	2	2	838
3	1	2	3	1	1	2	1	1	3	2	2	2	1	1	3	2	3	1	3	1	1	1	2	839
3	2	3	2	3	1	2	1	2	3	2	2	2	1	2	2	3	1	2	2	1	1	3	2	840
2	1	1	1	3	2	3	1	3	2	3	2	1	1	1	2	3	1	2	1	1	2	3	1	841
3	2	1	3	1	3	2	2	2	3	1	2	2	2	3	1	1	1	3	1	1	2	1	2	842
3	1	1	2	1	2	2	3	2	2	1	2	3	2	2	2	3	2	2	1	2	3	1	3	843
3	2	3	2	1	1	2	1	1	3	1	2	3	2	1	2	2	3	2	2	3	2	2	2	844
2	1	1	1	2	2	3	1	2	2	3	2	3	1	3	2	2	3	1	1	3	1	1	2	845
2	3	1	3	1	2	1	3	2	2	1	2	1	3	2	2	1	1	3	2	2	2	1	3	846
1	3	2	2	2	3	2	2	1	1	3	1	2	2	1	2	3	2	1	3	1	1	1	3	847
3	1	1	2	3	2	3	2	1	3	1	1	2	1	1	3	1	3	1	2	2	1	1	1	848
3	2	1	2	2	1	2	3	1	1	1	3	1	1	3	2	2	3	2	2	3	2	2	2	849
3	2	3	2	2	1	2	1	3	1	1	3	2	2	1	1	1	2	3	2	2	1	1	3	850
2	2	1	1	3	1	3	2	1	3	2	3	1	1	2	1	2	3	1	2	1	3	2	1	851
1	1	2	3	2	2	1	2	1	1	3	1	2	3	1	3	1	3	2	2	2	1	3	2	852
1	2	1	2	1	1	3	1	2	2	2	3	1	2	3	2	1	3	2	3	2	1	3	2	853
2	1	2	3	2	2	2	3	2	2	3	2	2	3	2	2	1	1	3	2	2	2	3	1	854
3	1	2	1	3	2	2	2	1	3	2	1	2	1	3	1	1	3	1	2	1	1	1	3	855
3	2	2	3	1	1	2	1	2	1	3	1	3	1	2	1	3	2	1	1	1	2	1	3	856
1	3	1	3	1	1	3	1	2	2	2	1	3	2	1	1	3	1	1	2	3	1	2	1	857
2	3	1	1	2	3	1	3	1	1	1	3	1	2	1	2	2	3	1	3	2	1	2	2	858
2	3	1	1	3	1	2	2	1	2	1	3	2	1	3	2	2	3	2	1	2	1	3	1	859
3	1	2	2	1	3	2	1	3	2	1	2	2	3	1	1	3	1	2	2	1	2	3	2	860
2	3	1	1	1	2	3	2	3	2	1	2	2	2	3	2	1	2	3	2	2	2	1	3	861
1	2	2	1	1	1	3	2	2	3	1	2	1	2	3	1	1	1	3	1	1	3	2	3	862
1	1	2	3	2	1	3	1	3	1	2	2	3	2	1	3	2	3	1	1	2	1	2	2	863
2	2	1	2	2	2	3	2	2	3	1	3	2	3	2	1	1	1	2	3	2	3	1	2	864
1	2	3	2	1	1	2	2	3	2	3	1	1	2	1	1	2	3	2	1	2	3	2	3	865
3	1	2	2	2	3	2	1	2	1	3	1	3	1	2	2	1	3	2	1	1	3	2	1	866
1	1	2	1	2	2	3	2	2	3	2	2	2	1	3	1	3	1	1	1	3	1	1	3	867
1	2	3	1	2	3	1	2	3	2	1	2	2	2	3	2	1	1	1	3	1	3	2	1	868
1	1	2	3	2	1	2	2	2	3	2	3	2	3	1	2	2	3	2	3	2	1	1	1	869
1	3	2	3	2	2	1	2	3	1	1	3	1	1	2	1	3	2	1	1	3	1	1	2	870
3	2	2	1	2	3	2	1	3	1	3	1	2	3	1	1	1	3	1	1	1	2	2	1	871
3	2	2	2	3	2	1	2	2	1	3	1	2	1	1	1	2	3	1	3	2	2	3	2	872
2	3	1	2	2	2	1	2	3	1	3	1	2	2	1	1	3	1	3	1	1	1	3	1	873
2	2	2	3	2	3	2	3	2	2	1	2	2	3	2	1	1	2	2	3	1	3	1	2	874
3	1	2	3	2	3	2	3	1	2	1	2	3	1	2	2	1	1	1	3	1	1	1	2	875
1	3	1	2	2	1	2	1	3	1	2	2	2	3	2	1	3	1	3	1	1	1	3	2	876
3	1	1	3	1	3	2	1	2	3	2	1	1	2	1	3	2	1	2	2	3	2	1	2	877
2	2	2	3	2	1	1	2	3	2	2	3	2	2	3	1	3	2	2	2	1	1	3	1	878
1	3	2	1	1	1	2	1	3	2	1	3	2	1	2	3	1	1	2	1	1	3	1	3	879
3	1	1	2	3	2	2	3	1	1	2	2	3	1	1	1	2	1	2	3	1	3	2	2	880
1	3	2	1	3	2	2	1	1	2	2	3	1	2	1	3	2	1	1	3	2	2	2	3	881
1	3	2	3	2	1	1	1	3	1	1	1	2	3	1	1	2	3	1	1	2	1	1	3	882
2	3	2	2	1	3	1	2	1	2	2	2	3	2	3	1	1	1	2	3	2	3	1	1	883
2	3	2	1	2	3	2	2	3	1	3	2	2	2	3	1	1	2	2	3	2	2	1	2	884
2	3	1	3	2	3	1	1	2	2	1	3	2	2	1	2	3	2	2	3	2	2	1	2	885
3	1	1	3	1	1	1	3	1	1	1	2	3	1	3	1	1	1	3	1	2	2	1	2	886
2	2	1	1	3	2	1	1	3	2	2	3	2	3	2	2	3	1	2	1	2	2	1	3	887
1	2	3	1	2	3	2	3	2	2	2	3	1	2	2	2	3	1	1	2	2	3	1	1	888
1	1	3	2	1	1	3	2	3	1	1	1	2	2	3	2	2	3	2	2	2	3	1	1	889
1	2	3	1	1	3	2	3	2	1	1	1	3	2	2	2	3	1	1	1	3	1	1	1	890
1	3	1	3	1	3	2	1	1	3	1	2	1	1	2	2	3	2	1	2	1	3	2	1	891
2	2	2	1	2	3	1	3	1	2	1	3	1	2	3	1	1	1	2	1	1	3	2	3	892
1	3	1	1	1	2	2	1	3	2	1	3	2	1	1	2	3	1	2	2	2	3	2	3	893
3	1	2	2	2	3	1	3	1	2	2	3	1	1	2	3	1	3	1	1	2	1	2	1	894
3	1	2	2	1	3	1	1	1	3	1	2	3	1	1	2	1	1	1	3	1	2	3	1	895
2	1	3	1	2	1	3	1	1	1	3	2	1	2	1	2	3	2	2	3	2	1	3	2	896
3	1	1	3	1	2	1	3	2	1	1	1	3	2	1	1	1	3	2	1	1	3	2	2	897
1	1	1	2	3	2	3	2	3	2	2	2	1	3	2	1	3	2	2	3	2	1	1	1	898
2	2	3	2	2	3	1	1	3	2	1	1	3	1	3	1	2	3	1	1	2	1	1	1	899
2	1	2	2	2	3	1	3	1	3	1	1	1	3	1	1	1	3	1	3	2	2	2	1	900
2	1	2	2	2	1	3	2	3	1	2	3	1	1	2	2	2	3	2	3	1	2	3	2	901
2	2	1	2	1	3	2	3	1	2	3	1	2	3	1	2	1	1	3	2	2	3	1	2	902
2	1	1	1	3	1	2	1	1	2	2	3	2	1	3	1	1	1	3	2	1	3	2	3	903
3	2	2	2	1	3	2	1	2	2	3	1	2	1	2	2	3	2	3	2	3	2	1	1	904
3	2	3	2	2	3	2	3	1	1	2	1	1	3	1	2	2	3	1	1	1	2	1	2	905
1	1	1	3	1	1	1	3	2	1	2	1	1	1	3	2	3	1	3	1	2	1	3	1	906
2	1	2	2	2	3	2	1	1	3	1	1	3	2	3	2	1	3	1	2	1	2	2	3	907
2	1	3	1	1	3	1	2	3	1	1	1	2	2	3	2	3	1	2	2	2	3	2	2	908
1	2	1	1	2	1	3	2	1	1	3	2	3	1	1	2	3	1	2	3	1	3	1	2	909
1	1	2	3	2	3	1	1	2	1	1	3	1	2	1	1	1	3	2	3	2	3	2	1	910
1	2	2	3	1	1	3	1	2	1	1	1	3	1	2	3	2	2	3	2	2	2	1	3	911
2	3	1	1	1	2	1	3	1	1	3	2	3	1	3	1	2	2	1	2	1	3	2	1	912
1	3	2	2	1	2	2	3	2	3	1	1	1	3	1	3	2	2	2	1	2	3	1	2	913
1	1	1	2	1	3	2	1	3	2	3	1	2	1	3	1	3	1	1	3	1	2	2	2	914
1	3	2	3	2	1	2	3	1	1	3	2	3	2	1	1	2	1	1	3	1	2	2	1	915
2	3	1	2	2	1	1	3	1	2	2	3	2	3	2	1	3	2	3	2	2	1	2	1	916
1	3	2	2	2	1	2	3	1	2	1	2	2	2	3	2	1	3	1	2	3	1	3	2	917
2	1	2	3	2	3	2	1	2	3	1	1	3	1	2	2	1	2	1	3	2	2	1	3	918
3	1	1	1	2	2	3	2	2	3	2	1	2	1	3	1	3	2	3	1	2	1	2	1	919
2	1	3	1	1	1	2	1	3	2	2	2	1	1	3	2	1	2	1	3	2	3	2	3	920
2	3	1	2	2	2	1	3	1	2	3	2	2	2	1	2	2	3	2	3	1	3	1	1	921
1	1	3	2	2	3	1	2	1	2	2	2	3	2	2	3	2	2	1	3	1	2	3	1	922
2	3	1	2	3	2	3	1	2	1	1	2	3	1	3	1	1	2	1	1	1	3	1	1	923
1	1	1	3	2	2	2	1	3	2	2	2	3	2	1	2	2	1	3	2	1	3	1	3	924
1	3	2	2	2	3	1	2	3	2	3	1	2	1	3	2	1	1	1	2	1	3	1	1	925
1	1	3	2	3	2	2	1	2	2	3	1	1	2	3	2	3	1	2	3	2	2	1	2	926
1	1	1	2	2	3	1	1	3	2	3	2	3	1	2	1	1	2	3	2	2	2	3	2	927
3	2	2	2	1	3	2	3	1	2	2	1	1	1	3	1	2	1	3	1	2	2	1	3	928
1	2	1	1	3	2	3	2	1	2	1	1	3	1	3	1	1	3	2	3	2	2	1	1	929
1	2	3	1	1	2	2	2	3	2	2	2	3	2	3	1	2	3	1	1	3	2	2	1	930
1	1	1	3	1	1	2	2	3	1	3	1	1	1	2	3	1	1	1	3	2	2	1	3	931
1	3	2	3	2	1	1	3	1	3	2	1	2	1	1	1	3	2	1	2	2	2	3	1	932
3	1	1	2	2	1	1	3	1	2	2	3	2	2	1	2	1	2	3	2	3	1	3	2	933
2	1	2	3	1	1	1	3	2	3	2	2	3	2	2	2	1	1	3	2	1	1	3	1	934
2	1	1	1	3	2	1	1	1	2	3	2	2	1	2	3	2	3	1	3	1	3	1	1	935
1	1	1	3	1	2	1	2	2	3	1	2	2	3	1	3	1	2	1	3	1	3	2	2	936
1	1	3	2	3	1	2	1	2	3	1	1	2	1	2	3	2	3	1	3	1	1	1	2	937
1	1	1	2	1	3	1	3	2	2	2	3	2	2	1	1	2	3	2	1	1	3	2	3	938
3	1	2	2	2	1	3	1	2	3	1	3	2	2	1	1	3	1	1	2	2	2	1	3	939
2	2	3	2	1	1	1	2	3	1	3	2	3	2	3	1	1	2	1	2	2	3	2	1	940
1	3	2	1	3	2	3	2	1	2	2	2	3	1	3	1	2	1	1	2	1	3	1	1	941
2	3	1	3	2	2	1	1	1	3	1	3	2	2	3	2	2	3	1	2	1	2	2	2	942
1	1	1	3	1	3	2	3	2	1	2	2	1	3	1	1	1	2	1	3	2	2	2	3	943
3	2	2	2	1	3	2	2	1	2	2	2	3	1	2	3	1	3	1	2	1	1	2	3	944
1	1	3	2	3	2	1	1	1	2	3	1	1	2	1	1	1	3	1	3	2	2	3	2	945
1	1	2	1	1	1	3	2	3	1	3	2	1	3	1	1	3	2	3	2	1	1	2	2	946
2	1	2	2	3	1	3	2	2	2	3	2	3	2	1	1	1	3	1	1	3	1	2	1	947
2	2	2	1	2	1	3	2	2	3	2	2	3	2	3	2	2	3	1	1	1	3	2	2	948
1	2	3	1	1	1	2	1	2	3	1	2	2	3	2	3	2	2	2	3	2	2	3	2	949
1	1	1	3	1	3	1	2	3	2	1	1	1	3	2	3	1	3	2	2	1	2	2	1	950
2	2	3	1	1	3	1	1	1	3	2	2	1	3	1	2	3	1	2	3	1	1	2	2	951
1	2	3	2	2	1	2	2	2	3	2	2	2	1	3	2	2	2	3	2	3	2	3	1	952
1	1	1	2	1	2	3	1	1	2	2	2	3	1	1	3	1	3	1	1	3	2	3	1	953
3	1	2	2	1	3	1	2	1	2	1	3	1	1	2	1	2	2	3	1	1	3	1	3	954
2	2	1	3	1	1	2	1	1	3	1	3	1	1	1	2	3	2	1	2	3	2	3	2	955
2	2	2	1	2	3	1	1	1	3	1	3	1	1	3	2	3	2	1	2	2	1	2	3	956
3	2	1	1	3	2	1	2	2	1	1	3	2	3	2	3	1	2	2	2	1	3	2	1	957
1	2	1	1	1	3	1	3	1	1	3	2	1	1	1	3	1	3	2	1	1	1	3	2	958
1	2	2	3	2	2	1	1	2	2	3	1	1	3	2	3	2	1	2	3	1	1	1	3	959
2	1	2	1	2	1	3	2	2	3	1	3	2	2	3	1	3	2	1	1	3	1	2	2	960
2	1	3	1	2	3	1	3	1	2	1	2	1	2	3	1	1	1	3	1	2	1	3	2	961
1	2	1	1	3	1	1	3	1	2	3	1	2	2	2	3	2	3	2	1	1	1	2	3	962
2	2	1	3	2	1	1	2	1	1	3	1	1	1	3	1	2	3	1	1	3	1	3	1	963
3	1	2	2	2	3	2	3	1	3	2	1	1	1	3	2	1	1	1	2	1	3	1	1	964
1	1	1	2	1	3	1	2	3	2	1	3	1	1	2	2	2	3	2	3	2	3	2	2	965
3	1	1	1	2	2	1	3	2	3	2	2	2	3	2	3	2	3	2	1	2	2	1	2	966
1	2	2	2	3	1	3	2	1	2	3	1	2	1	3	1	1	3	1	2	2	3	2	2	967
1	2	1	3	1	3	2	2	3	1	1	3	2	1	2	3	2	1	1	1	3	2	2	2	968
2	1	1	2	2	2	3	2	3	1	1	2	3	2	2	3	2	2	1	2	2	3	2	3	969
2	2	1	3	2	2	2	1	2	3	1	3	1	3	2	3	1	3	1	2	2	2	1	1	970
3	2	2	3	2	2	1	3	1	3	2	3	2	2	2	1	2	3	1	1	1	2	2	2	971
2	2	2	1	2	2	3	2	3	1	2	3	2	3	1	1	1	2	1	1	3	1	3	1	972
3	2	1	1	3	2	1	1	2	1	2	3	1	2	1	3	2	3	1	2	2	1	1	3	973
2	3	1	3	1	2	3	1	2	3	2	1	2	1	2	3	2	1	3	2	1	1	2	1	974
1	1	2	2	3	1	3	1	1	1	3	1	3	1	2	1	1	1	2	3	1	2	1	3	975
2	2	2	3	1	1	3	2	3	1	2	3	2	2	1	3	1	1	2	3	2	2	2	1	976
1	3	2	2	3	2	2	3	2	3	2	1	1	2	2	3	2	2	1	3	2	1	1	1	977
1	2	1	3	2	3	1	3	1	1	3	2	3	1	2	1	1	3	1	2	1	2	2	2	978
3	2	3	2	3	1	2	1	1	3	2	1	1	2	2	3	1	3	2	2	1	1	1	2	979
2	1	3	2	2	1	2	2	3	2	2	2	3	2	3	1	1	2	2	2	3	1	3	1	980
1	2	1	3	2	2	3	1	1	2	1	3	2	1	1	2	2	2	3	1	1	3	1	3	981
1	2	3	2	2	2	3	2	3	2	2	2	3	1	1	2	1	3	1	3	1	1	2	1	982
2	3	1	2	1	1	1	3	1	2	1	2	3	1	3	1	3	1	2	2	3	2	1	1	983
2	1	1	1	3	1	2	3	1	3	1	2	3	2	2	3	2	2	1	1	1	3	2	2	984
1	1	3	2	3	1	1	1	2	2	2	3	2	1	1	3	1	1	2	2	1	3	2	3	985
3	1	1	1	2	3	1	3	1	3	2	2	1	2	2	3	1	2	1	3	2	2	2	1	986
2	2	2	3	2	1	1	1	2	3	1	3	1	2	1	2	1	3	2	3	2	2	1	3	987
3	2	2	1	1	2	2	3	2	3	1	2	1	2	2	2	3	1	2	2	1	3	2	3	988
1	3	1	3	2	3	2	2	3	1	2	1	1	1	3	1	2	3	2	2	2	1	2	1	989
1	1	2	2	3	2	3	1	3	1	1	1	2	2	3	1	2	1	1	3	1	1	3	1	990
2	2	1	1	1	3	1	3	1	1	2	2	3	1	3	1	1	3	1	3	1	1	1	2	991
2	2	3	2	2	1	3	1	1	3	1	1	2	2	3	1	1	2	3	2	1	2	3	2	992
1	3	2	2	1	1	3	1	2	1	2	3	2	3	2	3	1	2	3	2	2	2	1	1	993
2	3	1	3	2	2	1	2	3	2	2	3	2	1	1	2	1	3	1	1	1	2	2	3	994
2	2	1	3	1	2	1	1	3	2	2	2	1	3	1	3	1	2	2	3	1	3	1	1	995
1	2	3	1	3	2	1	1	2	1	1	3	1	3	2	1	2	2	2	3	1	1	3	2	996
2	3	2	2	2	1	1	3	2	3	2	1	1	2	3	1	2	2	2	3	2	2	1	3	997
2	2	3	1	1	3	1	1	3	1	2	2	3	2	2	1	2	2	3	2	2	3	1	1	998
2	1	2	1	3	1	1	1	3	1	2	2	1	1	1	3	1	3	2	3	1	1	2	3	999
2	1	1	1	2	2	3	2	2	1	3	1	1	1	2	2	2	3	1	3	2	3	2	3	1000
1	2	2	3	2	2	1	3	2	3	2	3	2	2	1	2	2	3	1	2	2	1	2	3	1001
3	1	3	1	1	2	2	1	2	3	2	3	2	3	1	1	2	1	2	1	3	1	1	1	1002
2	2	3	1	2	2	3	1	2	1	1	1	3	2	1	1	1	3	1	3	2	3	2	1	1003
3	2	3	2	3	2	1	1	1	2	2	3	1	1	2	1	2	3	2	2	1	1	2	3	1004
1	1	1	3	2	1	1	1	3	1	1	1	3	1	1	3	2	2	2	3	1	1	1	3	1005
2	2	2	1	3	2	2	3	1	1	3	1	1	2	1	3	1	1	1	3	1	1	1	3	1006
3	2	3	2	1	1	2	1	1	3	1	3	2	3	1	1	2	1	3	2	1	1	2	2	1007
2	1	2	2	3	1	1	1	2	1	1	3	1	3	1	3	1	2	2	2	3	2	3	1	1008
1	2	3	1	3	1	1	1	3	1	1	3	1	1	3	2	2	1	1	3	1	2	2	2	1009
1	1	3	1	3	2	3	1	3	2	1	2	1	2	2	3	2	2	1	1	1	3	1	1	1010
2	2	2	3	2	1	1	1	3	2	3	1	2	3	1	2	3	2	1	1	3	1	2	1	1011
3	1	2	3	2	2	1	2	3	2	3	1	2	3	1	1	1	2	1	2	3	2	1	2	1012
3	2	1	3	1	1	2	1	1	1	3	2	3	2	2	1	1	1	3	2	3	2	2	1	1013
1	1	1	3	1	3	2	1	2	3	2	3	2	3	2	1	2	3	1	2	1	2	2	2	1014
1	1	1	3	1	2	1	1	3	1	3	2	2	1	3	2	1	1	1	2	2	3	2	3	1015
1	1	3	1	1	2	2	1	3	1	3	1	1	2	1	1	3	2	3	2	3	1	2	1	1016
3	1	2	1	1	3	1	1	1	3	2	3	1	1	1	2	3	2	1	1	1	2	2	3	1017
3	1	2	3	1	1	1	3	1	2	3	2	2	2	1	1	1	3	2	2	2	3	2	2	1018
1	3	2	3	2	1	1	3	2	1	1	2	1	1	3	2	2	2	3	1	3	1	1	1	1019
3	2	2	3	1	3	1	1	2	2	1	3	1	1	2	2	2	3	1	2	1	1	1	3	1020
2	2	1	1	3	1	1	1	2	2	2	3	2	1	2	3	2	3	2	2	3	2	2	3	1021
1	3	1	1	3	1	2	2	2	1	3	1	2	3	1	1	1	2	3	1	3	2	2	2	1022
2	1	1	3	2	2	2	3	1	3	1	2	1	1	1	3	1	2	3	1	2	1	2	3	1023
2	3	1	3	1	2	1	3	2	2	2	3	2	1	1	2	1	2	3	2	2	2	3	2	1024
1	3	2	2	2	3	1	1	1	2	2	3	2	1	1	3	2	2	2	3	1	2	3	1	1025
2	1	3	1	1	2	2	3	1	2	2	1	1	2	3	1	2	3	1	3	2	1	3	2	1026
1	3	1	3	1	2	2	2	3	2	1	1	2	1	1	3	2	1	2	2	3	1	1	3	1027
1	2	1	1	2	3	1	2	3	2	1	1	2	3	2	1	1	3	2	1	3	2	3	2	1028
2	3	1	1	1	2	2	2	3	1	2	3	1	3	1	3	1	2	1	2	3	2	2	1	1029
2	3	2	3	2	1	1	1	3	2	1	2	1	3	2	2	2	1	2	3	2	2	1	3	1030
2	3	1	1	2	1	1	3	2	3	1	1	1	2	1	3	1	1	2	3	1	1	2	3	1031
1	1	1	3	1	1	1	3	1	2	2	3	2	1	1	2	1	1	3	2	1	3	1	3	1032
1	1	2	3	1	1	1	2	1	3	2	3	2	2	1	1	1	2	3	1	3	2	3	2	1033
3	2	1	3	1	2	1	1	1	3	1	2	3	2	3	1	1	2	2	1	2	3	1	2	1034
3	1	2	1	3	2	1	2	1	2	3	2	3	2	3	2	1	2	2	2	3	2	2	2	1035
1	2	3	2	2	2	3	2	1	3	1	1	1	2	3	2	2	2	3	1	1	3	1	2	1036
1	1	1	2	2	2	3	2	1	3	1	3	1	3	1	1	1	2	2	2	3	2	2	3	1037
2	1	3	1	1	2	1	1	3	1	2	2	1	3	2	1	1	3	2	3	2	1	3	1	1038
2	3	1	2	2	2	1	3	1	3	1	1	1	2	1	2	3	1	3	2	1	3	1	1	1039
1	1	2	1	3	1	3	2	1	2	3	2	2	3	2	2	2	1	2	3	1	3	1	1	1040
3	1	2	3	1	2	3	1	1	3	1	3	2	2	2	1	2	2	3	2	1	1	1	2	1041
1	1	3	2	1	1	1	3	1	1	3	1	1	3	1	1	1	2	3	2	3	2	2	1	1042
2	2	3	1	1	3	1	1	2	2	1	1	3	2	3	2	2	2	1	3	2	3	2	1	1043
1	3	1	1	1	3	1	1	2	3	2	2	3	1	2	2	2	1	2	3	1	2	3	2	1044
3	1	2	2	1	1	1	3	1	3	1	2	3	2	2	3	1	2	2	3	1	1	1	2	1045
1	1	2	3	1	2	1	1	2	2	3	2	2	3	1	3	1	3	1	3	2	1	1	2	1046
3	2	2	2	3	2	2	3	1	1	1	3	2	3	2	1	1	1	3	2	1	2	1	2	1047
2	3	1	3	2	2	1	2	1	2	3	1	3	1	1	1	3	2	3	2	1	1	2	2	1048
2	2	3	2	3	1	3	1	1	1	3	1	1	3	2	1	2	1	2	1	3	1	1	2	1049
3	2	1	1	3	2	2	2	1	3	1	3	2	2	1	2	1	3	1	3	2	2	2	1	1050
3	1	2	1	3	1	2	1	3	1	2	1	1	3	2	2	1	1	2	2	3	1	1	3	1051
1	3	1	3	1	2	3	1	2	2	3	2	2	2	1	2	3	2	1	2	2	1	2	3	1052
1	1	1	3	2	2	1	1	3	1	1	1	2	2	3	2	1	3	2	3	1	2	1	3	1053
2	2	2	3	1	2	1	2	2	3	2	2	2	3	2	3	1	3	2	3	2	1	2	1	1054
1	2	2	2	3	2	1	3	1	1	1	3	2	2	3	2	2	1	2	3	1	3	2	2	1055
3	1	2	2	2	3	1	3	2	1	1	3	2	2	2	1	2	1	3	1	2	3	1	1	1056
1	1	3	1	2	1	1	1	3	2	3	1	3	2	2	3	1	2	2	2	1	3	1	2	1057
3	1	2	1	2	2	3	2	1	1	3	1	2	1	2	3	2	2	3	2	1	1	1	3	1058
3	2	1	1	3	1	3	2	3	2	1	2	2	3	2	1	1	3	2	2	1	1	2	2	1059
3	2	3	2	3	1	2	2	1	3	2	1	1	2	3	1	1	3	2	1	2	2	2	1	1060
3	2	1	1	3	1	1	1	3	1	2	2	1	1	3	2	3	2	2	1	3	2	1	1	1061
1	3	2	1	3	1	1	1	3	2	2	3	1	1	1	2	2	3	1	2	2	1	2	3	1062
2	1	1	3	1	3	1	1	3	2	2	3	1	3	2	1	1	2	3	2	1	2	2	2	1063
3	2	2	1	1	3	1	1	1	2	1	3	2	1	3	1	2	1	1	3	2	3	1	1	1064
2	1	1	3	2	1	1	1	2	2	3	1	1	1	3	2	3	2	1	2	1	3	2	3	1065
1	1	3	1	2	3	2	1	2	3	2	2	2	1	2	2	3	2	2	3	2	3	2	1	1066
1	2	2	2	1	3	1	1	2	1	2	1	3	2	3	1	1	3	1	3	1	2	1	3	1067
3	2	2	1	2	3	1	1	1	3	1	3	2	1	2	3	2	3	2	2	1	1	1	2	1068
2	1	2	2	1	2	3	2	3	1	1	3	1	1	3	1	1	2	3	1	2	2	1	3	1069
2	1	1	2	1	1	3	2	2	3	1	1	3	1	3	1	1	2	2	3	2	2	3	2	1070
2	3	1	2	3	2	2	2	3	1	2	3	2	1	1	2	2	3	2	2	1	1	1	3	1071
3	2	3	1	1	1	3	1	2	2	2	3	1	3	2	2	2	3	2	1	2	1	1	2	1072
1	3	1	3	1	1	2	1	2	1	3	1	2	2	3	1	3	1	2	2	2	3	2	2	1073
2	2	2	3	1	3	1	2	3	2	3	1	2	3	1	2	1	1	1	3	2	2	1	1	1074
3	2	2	3	2	1	1	1	2	2	3	2	1	3	2	1	1	1	3	1	1	3	2	1	1075
3	2	3	2	2	1	2	3	1	2	3	2	2	3	2	2	2	3	2	1	2	2	1	2	1076
1	2	2	1	2	2	3	2	3	2	1	3	1	2	3	2	1	2	2	1	1	3	1	3	1077
3	2	2	1	3	1	1	1	3	1	2	2	2	1	3	1	1	3	2	2	1	3	2	2	1078
2	2	3	2	3	2	1	2	2	1	1	3	1	3	1	3	2	3	1	1	1	2	1	2	1079
3	2	2	2	1	1	3	1	2	1	3	1	1	1	3	1	3	2	3	1	2	2	2	1	1080
1	1	2	3	1	3	1	1	1	2	1	3	1	2	1	3	2	2	1	2	2	3	2	3	1081
2	3	1	1	2	2	3	1	1	2	1	1	3	1	1	2	2	2	3	2	2	3	2	3	1082
1	1	2	1	1	3	1	2	2	3	1	1	2	2	1	3	2	3	1	3	2	1	1	3	1083
1	1	2	3	2	2	2	3	1	3	1	3	1	2	2	2	1	3	2	1	1	1	3	1	1084
1	3	2	2	2	1	3	1	1	2	1	3	1	1	1	2	3	2	3	2	2	2	3	1	1085
2	1	2	1	1	3	2	1	1	3	2	3	2	2	1	1	3	1	2	2	2	3	1	3	1086
3	2	1	3	2	3	1	1	2	1	1	3	2	2	1	3	2	3	2	2	1	1	2	1	1087
1	1	3	2	3	2	3	2	2	1	1	1	3	2	1	1	1	2	3	2	1	3	1	2	1088
1	3	1	3	1	2	3	2	2	2	1	2	3	2	2	3	2	3	1	1	2	2	1	1	1089
1	3	2	2	3	1	1	2	1	2	2	3	1	2	3	1	2	1	1	3	1	1	3	1	1090
2	3	1	1	2	3	2	3	1	3	1	2	3	2	2	2	1	3	1	1	2	1	1	2	1091
1	1	2	1	1	2	3	1	2	3	2	1	1	3	2	2	2	3	1	3	2	2	2	3	1092
1	1	1	3	1	3	2	3	1	1	2	1	3	1	1	1	2	1	1	3	1	3	1	1	1093
1	1	2	1	1	1	3	2	2	1	2	2	3	1	3	1	3	1	3	2	2	2	1	3	1094
1	3	2	1	3	2	3	2	2	3	2	1	3	2	2	2	1	3	2	1	2	1	2	1	1095
3	2	1	1	3	1	1	2	3	2	1	2	2	1	3	1	2	1	2	2	2	3	2	3	1096
3	1	2	1	1	1	2	3	2	2	2	3	1	2	1	1	1	3	2	1	3	2	2	3	1097
1	2	1	3	2	1	2	3	2	1	2	3	2	3	2	3	1	1	3	1	2	2	2	1	1098
1	2	3	1	1	2	3	2	1	3	1	3	2	3	1	2	2	1	3	2	2	2	1	1	1099
3	2	1	3	2	1	2	2	2	1	3	2	3	1	2	3	2	1	1	3	1	1	2	1	1100
1	3	1	1	2	2	3	2	1	2	2	3	1	1	3	1	1	3	1	1	2	1	2	3	1101
2	2	2	1	2	1	3	1	1	2	2	3	1	3	1	3	1	1	3	2	2	1	1	3	1102
1	1	1	3	2	1	3	2	1	3	1	3	1	2	2	2	3	1	3	1	1	2	2	1	1103
2	2	2	1	1	1	3	1	1	1	3	2	1	2	2	3	2	1	1	3	1	3	2	3	1104
1	1	1	2	2	3	1	3	1	1	1	3	2	3	1	1	2	3	1	1	3	2	2	2	1105
1	1	3	1	1	1	2	1	1	3	2	1	2	3	1	2	1	3	2	1	3	2	1	3	1106
1	2	2	2	3	1	1	2	2	3	2	1	2	2	3	2	1	3	2	2	2	3	2	3	1107
1	1	3	1	3	1	1	2	1	1	2	3	2	1	3	1	3	1	2	1	2	1	1	3	1108
2	3	2	3	2	1	1	2	1	3	2	2	3	2	2	1	1	2	3	1	3	2	1	1	1109
2	1	2	1	3	2	2	3	2	1	3	2	2	2	1	3	1	2	3	1	1	2	3	2	1110
1	2	2	3	2	3	2	2	1	3	1	1	2	3	1	2	3	2	2	1	1	2	1	3	1111
3	2	2	2	3	2	1	2	1	3	2	1	2	2	2	3	1	2	2	3	1	2	3	2	1112
1	3	1	3	2	1	1	1	3	2	1	2	3	1	3	2	2	1	2	3	1	1	2	1	1113
3	1	1	1	3	2	2	2	1	1	3	2	3	1	2	3	2	1	2	1	2	2	3	2	1114
2	2	1	1	1	2	3	1	2	1	1	1	3	1	3	2	1	3	2	3	1	1	3	2	1115
2	2	1	1	1	2	3	2	3	2	3	1	3	1	1	3	1	2	3	1	1	2	T	1	1116
1	2	2	2	3	2	1	2	1	1	1	3	2	3	1	1	3	1	1	3	1	3	1	1	1117
2	3	1	2	2	1	3	2	1	2	2	2	3	2	3	1	1	3	1	3	1	2	2	2	1118
2	2	2	3	1	1	2	3	1	1	1	2	2	3	1	2	3	1	2	1	3	1	2	3	1119
1	3	1	3	2	1	1	3	1	2	2	1	1	3	1	1	2	1	1	3	1	1	1	3	1120
1	2	2	3	1	1	2	2	3	1	3	1	1	3	2	3	1	1	3	2	1	1	1	2	1121
2	2	2	1	3	1	3	1	1	3	2	1	2	2	3	2	2	2	3	1	1	1	3	1	1122
2	1	1	1	3	2	3	1	1	1	3	1	2	2	2	3	1	1	1	2	3	1	2	3	1123
3	1	1	1	3	2	2	1	3	1	3	1	1	1	2	3	2	1	3	1	1	1	2	2	1124
3	2	3	1	1	2	1	1	2	3	1	1	3	1	1	3	2	2	1	2	3	2	2	1	1125
2	2	3	2	3	1	1	2	1	1	1	3	2	1	3	1	2	3	2	3	2	2	1	2	1126
2	2	1	2	1	2	3	1	2	1	2	3	1	3	2	2	2	3	2	3	2	2	3	1	1127
2	2	3	1	2	2	2	3	2	3	2	3	1	3	2	1	2	2	1	3	2	2	1	2	1128
1	1	1	3	2	3	1	2	2	1	1	3	2	2	1	3	2	2	2	3	1	3	1	2	1129
2	2	3	2	1	2	2	2	3	2	1	2	1	1	2	3	2	2	3	1	1	3	1	3	1130
3	2	2	2	3	1	1	1	2	2	1	3	2	3	2	3	1	3	1	1	1	2	1	2	1131
1	1	2	3	2	2	3	1	3	1	2	2	3	1	2	1	1	2	3	2	2	3	1	1	1132
2	1	3	2	1	3	2	1	3	2	1	2	2	3	2	2	3	2	1	1	2	1	1	3	1133
3	2	2	3	2	1	1	2	2	2	3	1	3	2	3	2	2	1	3	2	2	1	2	2	1134
2	3	1	1	2	1	2	3	1	2	1	3	2	2	1	3	2	1	1	2	2	3	2	3	1135
2	3	1	2	1	3	2	1	2	3	2	2	2	3	2	3	1	2	2	1	1	1	3	1	1136
3	1	2	3	2	1	2	1	1	1	3	1	3	2	1	2	3	2	2	1	2	1	1	3	1137
1	3	2	3	1	3	1	2	2	2	1	3	1	1	3	1	2	3	2	2	1	2	2	1	1138
1	2	3	1	3	1	1	2	2	2	3	2	2	1	1	1	3	1	3	1	1	1	3	2	1139
1	1	1	3	1	1	2	2	1	3	2	1	2	3	1	2	1	3	1	2	3	1	3	1	1140
2	1	3	1	3	2	2	3	2	1	2	1	3	2	2	2	1	2	1	3	2	2	3	1	1141
3	2	1	3	1	1	2	3	1	2	2	3	2	2	2	1	3	1	1	3	1	2	2	2	1142
3	2	2	2	1	2	3	2	2	2	3	1	3	1	1	3	1	3	2	2	1	2	2	2	1143
2	1	3	1	1	3	2	2	2	3	1	1	1	3	2	2	1	2	2	3	1	2	2	3	1144
3	1	2	3	1	1	3	1	3	2	1	2	2	2	3	2	2	1	2	1	2	3	2	1	1145
3	1	2	3	1	1	2	1	2	1	3	2	1	1	3	2	1	2	2	3	1	3	2	1	1146
2	1	3	2	3	1	2	3	1	1	1	2	2	2	3	1	3	1	2	1	3	1	2	1	1147
3	1	1	1	3	1	1	1	2	2	3	1	1	3	1	3	2	2	2	3	1	2	1	2	1148
1	2	2	2	3	1	3	2	1	2	2	2	3	2	3	2	1	2	2	3	1	1	2	3	1149
1	2	3	1	3	2	2	3	1	1	1	2	2	2	3	1	1	3	2	1	2	2	3	2	1150
2	2	1	1	2	1	3	2	3	1	3	1	3	1	3	2	1	2	1	2	3	2	1	1	1151
1	2	2	1	1	3	1	3	1	3	2	3	1	3	2	1	1	1	2	3	2	1	1	1	1152
1	1	3	1	1	2	1	3	1	2	3	1	3	1	2	2	1	3	1	1	1	2	1	3	1153
1	3	2	2	2	1	1	1	3	1	3	2	2	1	3	1	1	2	2	3	1	1	1	3	1154
3	2	1	1	3	1	2	2	2	3	2	2	3	1	1	2	1	1	1	3	1	1	3	1	1155
1	3	1	3	1	1	1	3	1	1	3	2	2	1	1	1	3	2	3	1	2	1	2	2	1156
2	1	1	2	1	3	1	3	1	1	3	1	3	1	2	3	2	1	2	3	1	1	2	1	1157
2	2	1	2	2	1	3	2	3	1	2	1	1	3	2	3	1	1	3	2	2	2	1	3	1158
1	2	1	1	2	3	2	1	1	1	3	1	2	3	1	3	2	2	2	1	2	3	1	3	1159
2	2	3	1	2	2	2	3	1	3	1	3	2	2	3	1	2	1	1	3	1	2	2	2	1160
1	2	3	1	2	2	1	2	2	3	2	3	2	3	2	1	3	1	1	2	2	1	3	1	1161
2	1	2	1	1	1	3	1	2	1	2	1	3	2	1	3	1	2	3	1	2	3	2	3	1162
2	2	2	1	3	2	2	3	1	3	1	2	3	1	1	3	2	2	1	2	2	1	3	1	1163
1	2	2	3	1	1	2	2	3	1	2	1	2	1	3	2	3	2	1	1	1	3	2	3	1164
3	1	1	3	1	1	1	3	1	2	2	1	2	2	3	2	1	2	2	3	1	3	2	2	1165
1	2	2	3	1	3	2	3	2	1	3	2	3	1	2	2	2	1	3	1	1	1	2	1	1166
1	1	2	1	1	1	3	2	3	2	2	2	1	1	3	1	3	2	1	3	1	3	2	1	1167
3	2	1	3	1	3	1	2	1	1	2	2	3	1	2	3	2	3	2	1	1	2	2	2	1168

In Table IIA, each of the numerals 1 to 3 (numeric identifiers) represents a nucleotide base and the pattern of numerals 1 to 3 of the sequences the above list corresponds to the pattern of nucleotide bases present in the oligonucleotides of Table II, which oligonucleotides have been found to be non-cross-hybridizing, as described further in the detailed examples. Each nucleotide base is selected from the group of nucleotide bases consisting of A, C, G, and T/U. A particularly preferred embodiment of the invention, in which a specific base is assigned to each numeric identifier is shown in Table II, below.

[0057]
In one broad aspect, the invention is a composition comprising molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on a group of sequences as specified by numeric identifiers set out in Table IIA. In the sequences, each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that:
- - for any pair of sequences of the set:
  - M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19, where:
  - M1 is the maximum number of matches for any alignment in which there are no internal indels;
  - M2 is the maximum length of a block of matches for any alignment;
  - M3 is the maximum number of matches for any alignment having a maximum score;
  - M4 is the maximum sum of the lengths of the longest two blocks of matches for
  - any alignment of maximum score; and
  - M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein:
    - the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D−eg)), wherein:
      
      for each of (i) to (iv):
      
      (i) m=6, mm=6, og=0 and eg=6,
      
      (ii) m=6, mm=6, og=5 and eg=1,
      
      (iii) m=6, mm=2, og=5 and eg=1, and
      
      (iv) m=6, mm=6., og=6 and eg=0,
      
      A is the total number of matched pairs of bases in the alignment;
      
      B is the total number of internal mismatched pairs in the alignment;
      
      C is the total number of internal gaps in the alignment; and.
      
      D is the total number of internal indels in the alignment minus
      
      the total number of internal gaps in the alignment; and
    - wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv).
[0078]

An explanation of the meaning of the parameters set out above is given in the section describing detailed embodiments.
[0079]
In another broad aspect the invention is a composition containing molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on a group of sequences as set out in Table IIA wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that:
- for any pair of sequences of the set:
  - M1≦19, M2≦17, M3≦21, M4≦18, and M5≦20, where:
    - M1 is the maximum number of matches for any alignment in which there are no internal indels;
    - M2 is the maximum length of a block of matches for any alignment;
    - M3 is the maximum number of matches for any alignment having a maximum score;
    - M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and
    - M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein
      
      the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:
      
      for each of (i) to (iv)
      
      (i) m=6, mm=6, og=0 and eg=6,
      
      (ii) m=6, mm=6, og=5 and eg=1,
      
      (iii) m=6, mm=2, og=S and eg=1, and
      
      (iv) m=6, mm=6, og=6 and eg=0,
      
      A is the total number of matched pairs of bases in the alignment;
      
      B is the total number of internal mismatched pairs in the alignment;
      
      C is the total number of internal gaps in the alignment; and
      
      D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and
      
      wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv).
[0098]
In another broad aspect, the invention is a composition comprising molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on a group of sequences set out in Table IIA wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that:
- for any pair of sequences of the set:
  - M1≦19, M2≦17, M3≦21, M4≦18, and M5≦20, where:
    - M1 is the maximum number of matches for any alignment in which there are n internal indels;
    - M2 is the maximum length of a block of matches for any alignment;
    - M3 is the maximum number of matches for any alignment having a maximum score;
    - M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and
    - M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score, wherein:
      
      the score of an alignment is determined according to the equation 3A−B−3C−D, wherein:
      
      A is the total number of matched pairs of bases in the alignment;
      
      B is the total number of internal mismatched pairs in the alignment;
      
      C is the total number of internal gaps in the alignment; and
      
      D is the total number of internal indels in the alignment minus
      
      the total number of internal gaps in the alignment; and
[0112]

In preferred aspects, the invention provides a composition in which, for the group of 24mer sequences in which 1=A, 2=T and 3=G, under a defined set of conditions in which the maximum degree of hybridization between a sequence and any complement of a different sequence of the group of. 24mer sequences does not exceed 30% of the degree of hybridization between said sequence and its complement, for all said oligonucleotides of the composition, the maximum degree of hybridization between an oligonucleotide and a complement of any other oligonucleotide of the composition does not exceed 50% of the degree of hybridization of the oligonucleotide and its complement.
[0113]

More preferably, the maximum degree of hybridization between a sequence and any complement of a different sequence does not exceed 30% of the degree of hybridization between said sequence and its complement, the degree of hybridization between each sequence and its complement varies by a factor of between 1 and up to 10, more preferably between 1 and up to 9, more preferably between 1 and up to 8, more preferably between 1 and up to 7, more preferably between 1 and up to 6, and more preferably between 1 and up to 5.
[0114]

It is also preferred that the maximum degree of hybridization between a sequence and any complement of a different sequence does not exceed 25%, more preferably does not exceed 20.%, more preferably does not exceed 15%, more preferably does not exceed 10%, more preferably does not exceed 5%.
[0115]

Even more preferably, the above-referenced defined set of conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37° C.
[0116]

In the composition, the defined set of conditions can include the group of 24mer sequences being covalently linked to beads.
[0117]

In a particular preferred aspect, for the group of 24mers the maximum degree of hybridization between a sequence and any complement of a different sequence does not exceed 15% of the degree of hybridization between said sequence and its complement and the degree of hybridization between each sequence and its complement varies by a factor of between 1 and up to 9, and for all oligonucleotides of the set, the maximum degree of hybridization between an oligonucleotide and a complement of any other oligonucleotide of the set does not exceed 20% of the degree of hybridization of the oligonucleotide and its complement.
[0118]

It is possible that each 1 is one of A, T/U, G and C; each 2 is one of A, T/U, G and C; and each 3 is one of A, T/U, G and C; and each of 1, 2 and 3 is selected so as to be different from all of the others of 1, 2 and 3. More preferably, 1 is A or T/U, 2 is A or T/U and 3 is G or C. Even more preferably, 1 is A, 2 is T/U, and 3 is G.
[0119]

In certain preferred composition, each of the oligonucleotides is from twenty-two to twenty-six bases in length, or from twenty-three to twenty-five, and preferably, each oligonucleotide is of the same length as every other said oligonucleotide.
[0120]

In a particularly preferred embodiment, each oligonucleotide is twenty-four bases in length.
[0121]

It is preferred that no oligonucleotide contains more than four contiguous bases that are identical to each other.
[0122]

It is also preferred that the number of G's in each oligonucleotide does not exceed L/4 where L is the number of bases in said sequence.
[0123]

For reasons described below, the number of G's in each said oligonucleotide is preferred not to vary from the average number of G's in all of the oligonucleotides by more than one. Even more preferably, the number of G's in each said oligonucleotide is the same as-every other said oligonucleotide. In the embodiment disclosed below in which oligonucleotides were tested, the sequence of each was twenty-four bases in length and each oligonucleotide contained 6 G's.
[0124]

It is also preferred that, for each nucleotide, there is at most six bases other than G between every pair of neighboring pairs of G's.
[0125]

Also, it is preferred that, at the 5′-end of each oligonucleotide at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence of the oligonculeotide is a G. Similarly, it is preferred, at the 3′-end of each oligonucleotide that at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence of the oligonucleotide is a G.
[0126]

It is possible to have sequence compositions that include one hundred and sixty said molecules, or that include one hundred and seventy said molecules, or that include one hundred and eighty said molecules, or that include one hundred and ninety said molecules, or that include two hundred said molecules, or that include two hundred and twenty said molecules, or that include two hundred and forty said molecules, or that include two hundred and sixty said molecules, or that include two hundred and eighty said molecules, or that include three hundred said molecules, or that include four hundred said molecules, or that include five hundred said molecules, or that include six hundred said molecules, or that include seven hundred said molecules, or that include eight hundred said molecules, or that include nine hundred said molecules, or that include one thousand said molecules.
[0127]

It is possible, in certain applications, for each molecule to be linked to a solid phase support so as to be distinguishable from a mixture containing other of the molecules by hybridization to its complement. Such a molecule can be linked to a defined location on a solid phase support such that the defined location for each molecule is different than the defined location for different others of the molecules.
[0128]

In certain embodiments, each solid phase support is a microparticle and each said molecule is covalently linked to a different microparticle than each other different said molecule.
[0129]
In another broad aspect, the invention is a composition comprising a set of 150 molecules for use as tags or tag complements wherein each molecule includes an oligonucleotide having a sequence of at least sixteen nucleotide bases wherein for any pair of sequences of the set:
- - M1≦19/24×L1, M2≦17/24×L1, M3≦21/24×L1, M4≦18/24×L1,
  - M5≦20/24×L1, where L1 is the length of the shortest sequence of the pair, where:
    - M1 is the maximum number of matches for any alignment of the pair of sequences in which there are no internal indels;
    - M2 is the maximum length of a block of matches for any alignment of the pair of sequences;
    - M3 is the maximum number of matches for any alignment of the pair of sequences having a maximum score;
    - M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of the pair of sequences of maximum score; and
    - M5 is the maximum sum of the lengths of all the blocks of matches having length of at least 3, for any alignment of the pair of sequences of maximum score, wherein:
      
      the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:
      
      for each of (i) to (iv):
      
      (i) m=6, mm=6, og=0 and eg=6,
      
      (ii) m=6, mm=6, og=5 and eg=1,
      
      (iii) m=6, mm=2, og=5 and eg=1, and
      
      (iv) m=6, mm=6, og=6 and eg=0,
      
      A is the total number of matched pairs of bases in the alignment;
      
      B is the total number of internal mismatched pairs in the alignment;
      
      C is the total number of internal gaps in the alignment; and
      
      D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and
      
      wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv).
[0148]
In yet another broad aspect, the invention is a composition that includes a set of 150 molecules for use as tags or tag complements wherein each molecule has an oligonucleotide having a sequence of at least sixteen nucleotide bases wherein for any pair of sequences of the set:
- - M1≦19, M2≦17, M3≦21, M4≦18, and M5≦20, where:
    - M1 is the maximum number of matches for any alignment of the pair of sequences in which there are no internal indels;
    - M2 is the maximum length of a block of matches for any alignment of the pair of sequences;
    - M3 is the maximum number of matches for any alignment of the pair of sequences having a maximum score;
    - M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of the pair of sequences of maximum score; and
    - M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of the pair of sequences of maximum score, wherein:
      
      the score of a said alignment is determined according to the equation 3A−B−3C−D, wherein:
      
      A is the total number of matched pairs of bases in the alignment;
      
      B is the total number of internal mismatched pairs in the alignment;
      
      C is the total number of internal gaps in the alignment; and
      
      D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment.
[0160]

In certain embodiments of the invention., each sequence of a composition has up to fifty bases. More preferably, however, each sequence is between sixteen and forty bases in length, or between sixteen and thirty-five bases in length, or between eighteen and thirty bases in length, or between twenty and twenty-eight bases in length, or between twenty-one and twenty-seven bases in length, or between twenty-two and twenty-six bases in length.
[0161]

Often, each sequence is of the same length as every other said sequence. In particular embodiments disclosed-herein, each sequence is twenty-four bases in length.
[0162]

Again, it can be preferred that no sequence contains more than four contiguous bases that are identical to each other, etc., as described above.
[0163]

In certain preferred embodiments, the composition is such that, under a defined set of conditions, the maximum degree of hybridization between an oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed about 30% of the degree of hybridization between said oligonucleotide and its complement, more preferably 20%, more preferably 15%, more preferably 10%, more preferably 6%.
[0164]

Preferably, the set of conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37° C., and the oligonucleotides are covalently linked to microparticles. Of course it is possible that these specific conditions be used for determining the level of hybridization.
[0165]

It is also preferred that under such a defined set of conditions, the degree of hybridization between each oligonucleotide and its complement varies by a factor of between 1 and up to 8, more preferably up to 7, more preferably up to 6, more preferably up to 5. In a particular disclosed embodiment, the observed variance in the degree of hybridization was a factor of only 5.3, i.e., the degree of hybridization between each oligonucleotide and its complement varied by a factor of between 1 and 5.6.
[0166]

In certain preferred embodiments, under the defined set of conditions, the maximum degree of hybridization between a said oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed about 15%, more preferably 10%, more preferably 6%.
[0167]

In one preferred embodiment, the set of conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37° C., and the oligonucleotides are covalently linked to microparticles.
[0168]

Also, under the defined set of conditions, it is preferred that the degree of hybridization between each oligonucleotide and its complement varies by a factor of between 1 and up to 8, more preferably up to 7, more preferably up to 6, more preferably up to 5.
[0169]

Any composition of the invention can include one hundred and sixty of the oligonucleotide molecules, or one hundred and seventy of the oligonucleotide molecules, or one hundred and eighty of the oligonucleotide molecules, or one hundred and ninety of the oligonucleotide molecules, or two hundred of the oligonucleotide molecules, or two hundred and twenty of the oligonucleotide molecules, or two hundred and forty of the oligonucleotide molecules, or two hundred and sixty of the oligonucleotide molecules, or two hundred and eighty of the oligonucleotide molecules, or three hundred of the oligonucleotide molecules, or four hundred of the oligonucleotide molecules, or five hundred of the oligonucleotide molecules, or six hundred of the oligonucleotide molecules, or seven hundred of the oligonucleotide molecules, or eight hundred of the oligonucleotide molecules, or nine hundred of the oligonucleotide molecules, or one thousand or more of the oligonucleotide molecules.
[0170]

A composition of the invention can be a family of tags, or it can be a family of tag complements.
[0171]

An oligonucleotide molecule belonging to a family of molecules of the invention can have incorporated thereinto one more analogues of nucleotide bases, preference being given those that undergo normal Watson-Crick base pairing.
[0172]

The invention includes kits for sorting and identifying polynucleotides. Such a kit can include one or more solid phase supports each having one or more spatially discrete regions, each such region having a uniform population of substantially identical tag complements covalently attached. The tag complements are made up of a set of oligonucleotides of the invention.
[0173]

The one or more solid phase supports can be a planar substrate in which the one or more spatially discrete regions is a plurality of spatially addressable regions.
[0174]

The tag complements can also be coupled to microparticles. Microparticles preferably each have a diameter in the range of from 5 to 40 μm.
[0175]

Such a kit preferably includes microparticles that are spectrophotometrically unique, and therefore distinguisable from each other according to conventional laboratory techniques. Of course for such kits to work, each type of microparticle would generally have only one tag complement associated with it, and usually there would be a different oligonucleotide tag complement associated with (attached to) each type of microparticle.
[0176]

The invention includes methods of using families of oligonucleotides of the invention.
[0177]
One such method is of analyzing a biological sample containing a biological sequence for the presence of a mutation or polymorphism at a locus of the nucleic acid. The method includes:
- (A) amplifying the nucleic acid molecule in the presence of a first primer having a 5′-sequence having the sequence of a tag complementary to the sequence of a tag complement belonging to a family of tag complements of the invention to form an amplified molecule with a 5′-end with a sequence complementary to the sequence of the tag;
- (B) extending the amplified molecule in the presence of a polymerase and a second primer having 5′-end complementary the 3′-end of the amplified sequence, with the 3′-end of the second primer extending to immediately adjacent said locus, in the presence of a plurality of nucleoside triphosphate derivatives each of which is: (i) capable of incorporation during transciption by the polymerase onto the 3′-end of a growing nucleotide strand; (ii) causes termination of polymerization; and (iii) capable of differential detection, one from the other, wherein there is a said derivative complementary to each possible nucleotide present at said locus of the amplified sequence;
- (C) specifically hybridizing the second primer to a tag complement having the tag complement sequence of (A); and
- (D) detecting the nucleotide derivative incorporated into the second primer in (B) so as to identify the base located at the locus of the nucleic acid.
[0182]
In another method of the invention, a biological sample containing a plurality of nucleic acid molecules is analyzed for the presence of a mutation or polymorphism at a locus of each nucleic acid molecule, for each nucleic acid molecule. This method includes steps of:
- (A) amplifying the nucleic acid molecule in the presence of a first primer having a 5′-sequence having the sequence of a tag complementary to the sequence of a tag complement belonging to a family of tag complements of the invention to form an amplified molecule with a 5′-end with a sequence complementary to the sequence of the tag;
- (B) extending the amplified molecule in the presence of a polymerase and a second primer having 5′-end complementary the 3′-end of the amplified sequence, the 3′-end of the second primer extending to immediately adjacent said locus, in the presence of a plurality of nucleoside triphosphate derivatives each of which is: (i) capable of incorporation during transciption by the polymerase onto the 3′-end of a growing nucleotide strand; (ii) causes termination of polymerization; and (iii) capable of differential detection, one from the other, wherein there is a said derivative complementary to each possible nucleotide present at said locus of the amplified molecule;
- (C) specifically hybridizing the second primer to a tag complement having the tag complement sequence of (A); and
- (D) detecting the nucleotide derivative incorporated into the second primer in (B) so as to identify the base located at the locus of the nucleic acid;
  wherein each tag of (A) is unique for each nucleic acid molecule and steps (A) a:
- (B) are carried out with said nucleic molecules in the presence of each other.
[0188]
Another method includes analyzing a biological sample that contains a plurality of double stranded complementary nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each nucleic acid molecule, for each nucleic acid molecule. The method includes steps of:
- (A) amplifying the double stranded molecule in the presence of a pair of first primers, each primer having an identical 5′-sequence having the sequence of a tag complementary to the sequence of a tag complement belonging to a family of tag complements of the invention to form amplified molecules with 5′-ends with a sequence complementary to the sequence of the tag;
- (B) extending the amplified molecules in the presence of a polymerase and a p of second primers each second primer having a 5′-end complementary a 3′-end of the amplified sequence, the 3′-end of each said second primer extending to immediately adjacent said locus, in the presence of a plurality of nucleoside triphosphate derivatives each of which is: (i) capable of incorporation during transciption by the polymerase onto the 3′-end of a growing nucleotide strand; (ii) causes termination of polymerization; and (iii) capable of differential detection, one from the other;
- (C) specifically hybridizing each of the second primers to a tag complement having the tag complement sequence of (A); and
- (D) detecting the nucleotide derivative incorporated into the second primers in (B) so as to identify the base located at said locus;
  wherein the sequence of each tag of (A) is unique for each nucleic acid molecule and steps (A) and (B) are carried out with said nucleic molecules in the presence of each other.
[0193]
In yet another aspect, the invention is a method of analyzing a biological sample containing a plurality of nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each nucleic acid molecule, for each nucleic acid molecule, the method including steps of:
- (a) hybridizing the molecule and a primer, the primer having a 5′-sequence having the sequence of a tag complementary to the sequence of a tag complement belonging to a family of tag complements of the invention and a 3′-end extending to immediately adjacent the locus;
- (b) enzymatically extending the 3′-end of the primer in the presence of a plurality of nucleoside triphosphate derivatives each of which is: (i) capable of enzymatic incorporation onto the 3′-end of a growing nucleotide strand; (ii) causes termination of said extension; and (iii) capable of differential detection, one from the other, wherein there is a said derivative complementary to each possible nucleotide present at said locus;
- (c) specifically hybridizing the extended primer formed in step (b) to a tag complement having the tag complement sequence of (a); and
- (d) detecting the nucleotide derivative incorporated into the primer in step (b) so as to identify the base located at the locus of the nucleic a molecule;
  wherein each tag of (a) is unique for each nucleic acid molecule and steps (a) a (b) are carried out with said nucleic molecules in the presence of each other.
[0198]

The derivative can be a dideoxy nucleoside triphosphate.
[0199]

Each respective complement can be attached as a uniform population of substantially identical complements in spacially discrete regions on one or more solid phase support(s).
[0200]

Each tag complement can include a label, each such label being different for respective complements, and step (d) can include detecting the presence of the different labels for respective hybridization complexes of bound tags and tag complements.
[0201]
Another aspect of the invention includes a method of determining the presence of a target suspected of being contained in a mixture. The method includes the steps of:
- (i) labelling the target with a first label;
- (ii) providing a first detection moiety capable of specific binding to the target and including a first tag;
- (iii) exposing a sample of the mixture to the detection moiety under conditions suitable to permit (or cause) said specific binding of the molecule and target;
- (iv) providing a family of suitable tag complements of the invention wherein the family contains a first tag complement having a sequence complementary to that of the first tag;
- (v) exposing the sample to the family of tag complements under-conditions suitable to permit (or cause) specific hybridization of the first tag and its tag complement;
- (vi) determining whether a said first detection moiety hybridized to a first s tag complement is bound to a said labelled target in order to determine t presence or absence of said target in the mixture.
[0208]

Preferably, the first tag complement is linked to a solid support at a specific location of the support and step (vi) includes detecting the presence of the first label at said specified location.
[0209]

Also, the first tag complement can include a second label and step (vi) includes detecting the presence of the first and second labels in a hybridized complex of the moiety and the first tag complement.
[0210]

Further, the target can be selected from the group consisting of organic molecules, antigens, proteins, polypeptides, antibodies and nucleic acids. The target can be an antigen and the first molecule can be an antibody specific for that antigen.
[0211]

The antigen is usually a polypeptide or protein and the labelling step can include conjugation of fluorescent molecules, digoxigenin, biotinylation and the like.
[0212]

The target can be a nucleic acid and the labelling step can include incorporation of fluorescent molecules, radiolabelled nucleotide, digoxigenin, biotinylation and the like.
[0213]

Another aspect of the invention includes detecting the presence of a target nucleic acid molecule using the Invader Assay, which is described in detail in U.S. Pat. No. 5,985,557 issued Nov. 16, 1999, incorporated herein by reference. The sequences of the present invention are incorporated into the 3′ portion of one of the two oligonucleotide probes that will eventually be cleaved by a Cleavase enzyme and captured by its complement which may be attached on a solid phase support in a microarray format.
[0214]

Another aspect of the invention includes a method of analyzing a biological sample comprising a plurality of target nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule using the Invader Assay. Again, the sequences of the present invention are incorporated into the 3′ portion of one of the two probes that will eventually be cleaved by a Cleavase enzyme and detected by using the cleaved sequence's complement, which may be attached on a solid phase support such as in a microarray format.
[0215]

Another aspect of the invention incorporates the use of a second target nucleic acid sequence, wherein the second target nucleic acid sequence comprises a synthetic nucleic acid. The synthetic nucleic acid may further comprise at least one hairpin loop. The construction and use of such nucleic acid sequences with hairpin loops has been described in detail in U.S. Pat. No. 5,770,365 issued Jun. 23, 1998 and International Publication WO 01/94625A2 published Dec. 13, 2001.
[0216]

The present invention capitalizes on the exquisite specificity of the Invader Assay and the minimally cross-hybridizing sequences of the present invention such that simultaneous use of multiple hybridization probes in a single experiment is now possible. The methods and compositions of the present invention allow for accurate and homogenous genotyping of a plurality of distinct nucleic acid in a single experiment. The methods and compositions of the present invention are flexible enough to extend to novel loci with little optimization the features of both the Invader Assay and the sequences of the present invention lend the technology to automation.

DETAILED DESCRIPTION OF THE INVENTION

[0000]

Figures
[0217]

Reference is made to the attached figures in which,
[0218]

FIGS. 1A and 1B illustrate results obtained in the cross-hybridization experiments described in Example 1. FIG. 1A shows the hybridization pattern found when a microarray containing all 100 probes (SEQ ID NOs:1 to 100 of Table I) was hybridized with a 24mer oligonucleotide having the complementary sequence to SEQ ID NO:3 of Table I(target). FIG. 1B shows the pattern observed when a similar array was hybridized with a mix of all 100 targets, i.e., oligonucleotides having the sequences complementary to SEQ ID NOs:1 to 100 of Table 1.
[0219]

FIG. 2 shows the intensity of the signal (MFI) for each perfectly matched sequence (indicated in Table I) and its complement obtained as described in Example 3.
[0220]

FIG. 3 is a three dimensional representation showing cross-hybridization observed for the sequences of FIG. 2 as described in Example 3. The results shown in FIG. 2 are reproduced along the diagonal of the drawing.
[0221]

FIG. 4 is illustrative of results obtained for an individual target (SEQ ID NO:23 of Table I, target No. 16) when exposed to the 100 probes of Example 3. The MFI for each bead is plotted.
[0222]

FIG. 5 illustrates generally the steps followed to obtain a family of sequences of the present invention;
[0223]

FIG. 6 shows the intensity of the signal (MFI) for each perfectly matched sequence (probe sequence indicated in Table II) and its complement (target at 50 fmol) obtained as described in Example 4;
[0224]

FIG. 7 is a three dimensional representation showing cross-hybridization observed for the sequences of FIG. 6 as described in Example 4. The results shown in FIG. 6 are reproduced along the diagonal of the drawing;
[0225]

FIG. 8 is illustrative of the results obtained for an individual target (Table II, SEQ ID No: 90, target No. 90) when exposed to the 100 probes of Example 4. The MFI for each bead is plotted.

DETAILED DESCRIPTION OF THE INVENTION

[0226]

The invention provides a method for sorting complex mixtures of molecules by the use of families of oligonucleotide sequence tags. The families of oligonucleotide sequence tags are designed so as to provide minimal cross hybridization during the sorting process. Thus any sequence within a family of sequences will not cross hybridize with any other sequence derived from that family under appropriate hybridization conditions known by those skilled in the art. The invention is particularly useful in highly parallel processing of analytes.
[0000]

Families of Oligonucleotide Sequence Tags
[0227]

The present invention includes a family of 24mer polynucleotides, that have been demonstrated to be minimally cross-hybridizing with each other. This family of polynucleotides is thus useful as a family of tags, and their complements as tag complements.
[0228]

The oligonucleotide sequences that belong to families of sequences that do not exhibit cross hybridization behavior can be derived by computer programs (described in U.S. Provisional Patent Application No. 60/181,563 filed Feb. 10, 2000). The programs use a method of generating a maximum number of minimally cross-hybridizing polynucleotide sequences that can be summarized as follows. First, a set of sequences of a given length are created based on a given number of block elements. Thus, if a family of polynucleotide sequences 24 nucleotides (24mer) in length is desired from a set of 6 block elements, each element comprising 4 nucleotides, then a family of 24mers is generated considering all positions of the 6 block elements. In this case, there will be 6⁶(46,656) ways of assembling the 6 block elements to generate all possible polynucleotide sequences 24 nucleotides in length.
[0229]

Constraints are imposed on the sequences and are expressed as a set of rules on the identities of the blocks such that homology between any two sequences will not exceed the degree of homology desired between these two sequences. All polynucleotide sequences generated which obey the rules are saved. Sequence comparisons are performed in order to generate an incidence matrix. The incidence matrix is presented as a simple graph and the sequences with the desired property of being minimally cross hybridizing are found from a clique of the simple graph, which may have multiple cliques. Once a clique containing a suitably large number of sequences is found, the sequences are experimentally tested to determine if it is a set of minimally cross hybridizing sequences. This method has been used to obtain the 100 non cross-hybridizing tags of Table I that are the subject of Example 1.
[0230]

The method includes a rational approach to the selection of groups of sequences that are used to describe the blocks. For example there are n⁴different tetramers that can be obtained from n different nucleotides, non-standard bases or analogues thereof. In a more preferred embodiment there are 44 or 256 possible tetramers when natural nucleotides are used. More preferably 81 possible tetramers when only 3 bases are used A, T and G. Most preferably 32 different tetramers when all sequences have only one G.
[0231]

Block sequences can be composed of a subset of natural bases most preferably A, T and G. Sequences derived from blocks that are deficient in one base possess useful characteristics, for example, in reducing potential secondary structure formation or reduced potential for cross hybridization with nucleic acids in nature. Sets of block sequences that are most preferable in constructing families of non cross hybridizing tag sequences should contribute approximately equivalent stability to the formation of the correct duplex as all other block sequences of the set. This should provide tag sequences that behave isothermally. This can be achieved, for example, by maintaining a constant base composition for all block sequences such as one G and three A's or T's for each block sequence. Preferably, non-cross hybridizing sets of block sequences will be comprised from blocks of sequences that are isothermal. The block sequences should be different from each other by at least one mismatch. Guidance for selecting such sequences is provided by methods for selecting primer and or probe sequences that can be found in published techniques (Robertson et al., Methods Mol Biol; 98:121-54 (1998); Rychlik et al, Nucleic Acids Research, 17:8543-8551 (1989); Breslauer et al., Proc Natl Acad. Sci., 83:3746-3750 (1986)) and the like. Additional sets of sequences can be designed by extrapolating on the original family of non cross hybridizing sequences by simple methods known to those skilled in the art.
[0232]

A preferred family of 100 tags is shown as SEQ ID NOs:1 to 100 in Table I. Characterization of the family of 100 sequence tags was performed to determine the ability of these sequences to form specific duplex structures with their complementary sequences and to assess the potential for cross hybridization. The 100 sequences were synthesized and spotted onto glass slides where they were coupled to the surface by amine linkage. Complementary tag sequences were Cy3-labeled and hybridized individually to the array containing the family of 100 sequence tags. Formation of duplex structures was detected and quantified for each of the positions on the array. Each of the tag sequences performed as expected, that is the perfect match duplex was formed in the absence of significant cross hybridization under stringent hybridization conditions. The results of a sample hybridization are shown in FIG. 1. FIG. 1 a shows the hybridization pattern seen when a microarray containing all 100 probes was hybridized with the target complementary to probe 181234. The 4 sets of paired spots correspond to the probe complementary to the target. FIG. 1 b shows the pattern seen when a similar array was hybridized with a mix of all 100 targets. These results indicate that the family of sequences which is the subject of this patent can be used as a family of non-cross hybridizing (tag) sequences.
[0233]

The family of 100 non-cross-hybridizing sequences can be expanded by incorporating additional tetramer sequences that are used in constructing further 24mer oligonucleotides. In one example, four additional words were included in the generation of new sequences to be considered for inclusion as non-cross talkers in a family of sequences that were obtained from the above method using 10 tetramers. In this case, the four additional words were selected to avoid potential homologies with all potential combinations of other words: YYXW (TTAG); WYYX (GTTA); XYXW (ATAG) and WYYY (GTTT). The total number of sequences containing six words using the 14 possible words is 14⁶or 7,529,536. These sequences were screened to eliminate sequences that contain repetitive regions that present potential hybridization problems such as four or more of a similar base (e.g., AAAA or TTTT) or pairs of G's. Each of these sequences was compared to the sequence set of the original family of 100 non-cross-hybridizing sequences (SEQ ID NOs:1 to 100). Any new sequence that contained a minimal threshold of homology (that does not include the use of insertions or deletions) such as 15 or more matches with any of the original family of sequences was eliminated. In other words, if it was possible to align a new sequence with one or more of the original 100 sequences so as to obtain a maximum simple homology of 15/24 or more, the new sequence was dropped. “Simple homology” between a pair of sequences is defined here as the number of pairs of nucleotides that are matching (are the same as each other) in a comparison of two aligned sequences divided by the total number of potential matches. “Maximum simple homology” is obtained when two sequences are aligned with each other so as to have the maximum number of paired matching nucleotides. In any event, the set of new sequences so obtained was referred to as the “candidate sequences”. One of the candidate sequences was arbitrarily chosen and referred togas sequence 101. All the candidate sequences were checked against sequence 101, and sequences that contained 15 or more non-consecutive matches (i.e., a maximum simple homology of 15/24 (62.5%) or more were eliminated. This results in a smaller set of candidate sequences from which another sequence is selected that is now referred to as sequence 102. The smaller set of candidate sequences is now compared to sequence 102 eliminating sequences that contained 15 or more non-consecutive matches and the process is repeated until there are no candidate sequences remaining. Also, any sequence selected from the candidate sequences is eliminated if it has 13 or more consecutive matches with any other previously selected candidate sequence.
[0234]

The additional set of 73tag sequences so obtained (SEQ ID NOs:101 to 173 of Table 1) is composed of sequences that when compared to any of SEQ ID NOs:1 to 100 of Table I have no greater similarity than the sequences of the original 100 sequence tags of Table I. The sequence set as derived from the original family of non cross hybridizing sequences, SEQ ID NOs:1 to 173 of Table 1, are expected to behave with similar hybridization properties to the sequences having SEQ ID NOs:1 to 100 since it is understood that sequence similarity correlates directly with cross hybridization (Southern et al., Nat. Genet.; 21, 5-9: 1999).
[0235]

The set of 173 24mer oligonucleotides were expanded to include those having SEQ ID NOs:174 to 210 as follows. The 4mers WXYW, XYXW, WXXW, WYYW, XYYX, YXYX, YXXY and XYXY where W=G, X=A, and Y=U/T were used in combination with the fourteen 4mers used in the generation of SEQ ID NOs:1 to 173 to generate potential 24-base oligonucleotides. Excluded from the set were those containing the sequence patterns GG, AAAA and TTTT. To be included in the set of additional 24mers, a sequence also had to have at least one of the 4mers containing two G's: WXYW (GATG), WYXW (GTAG), WXXW (GAAG), WYYW (GTTG) while also containing exactly six G's. Also required for a 24mer to be included was that there be at most six bases between every neighboring pair of G's. Another way of putting this is that there are at most six non-G's between any two G's. Also, each G nearest the 5′-end of its oligonucleotide (the left-hand side as written in Table I) was required to occupy one of the first to seventh positions (counting the 5′-terminal position as the first position.) A set of candidate sequences was obtained by eliminating any new sequence that was found to have a maximum simple homology of 16/24 or more with any of the previous set of 173 oligonucleotides (Table 1, SEQ ID NOs:1 to 0.173). As above, an arbitrary 174^thsequence was chosen and candidate sequences eliminated by comparison therewith. In this case the permitted maximum degree of simple homology was 16/24. A second sequence was also eliminated if there were ten consecutive matches between the two (i.e., it was notionally possible to generate a phantom sequence containing a sequence of 10 bases that is identical to a sequence in each of the sequences being compared). A second sequence was also eliminated if it was possible to generate a phantom sequence 20 bases in length or greater.
[0236]

A property of the polynucleotide sequences shown in Table I is that the maximum block homology between any two sequences is never greater than 66⅔ percent. This is because the computer algorithm by which the sequences were initially generated was designed to prevent such an occurrence. It is within the capability of a person skilled in the art, given the family of sequences of Table I, to modify the sequences, or add other sequences while largely retaining the property of minimal-cross hybridization which the polynucleotides of Table I have been demonstrated to have.
[0237]

There are 210 polynucleotide sequences given in Table I. Since all 210 of this family of polynucleotides can work with each other as a minimally cross-hybridizing set, then any plurality of polynucleotides that is a subset of the 210 can also act as a minimally cross-hybridizing set of polynucleotides. An application in which, for example, 30 molecules are to be sorted using a family of polynucleotide tags and tag complements could thus use any group of 30 sequences shown in Table I. This is not to say that some subsets may be found in practical sense to be more preferred than others. For example, it may be found that a particular subset is more tolerant of a wider variety of conditions under which hybridization is conducted before the degree of cross-hybridization becomes unacceptable.
[0238]

It may be desirable to use polynucleotides that are shorter in length than the 24 bases of those in Table I. A family of subsequences (i.e., subframes of the sequences illustrated) based on those contained in Table I having as few as 10 bases per sequence could be chosen, so long as the subsequences are chosen to retain homological properties between any two of the sequences of the family important to their non cross-hybridization.
[0239]

The selection of sequences using this approach would be amenable to a computerized process. Thus for example, a string of 10 contiguous bases of the first 24mer of Table I could be selected: GATTTGTATTGATTGAGATTAAAG.
[0240]

A string of contiguous bases from the second 24mer could then be selected and compared for maximum homology against the first chosen sequence: TGATTGTAGTATGTATTGATAAAG

[0241]

Systematic pairwise comparison could then be carried out to determine if the maximum homology requirement of 66⅔ percent is violated:



	Alignment	Matches


	GATTTGTATT	1

	ATTGATAAAG

	GATTTGTATT	0

	ATTGATAAAG

	GATTTGTATT	1

	ATTGATAAAG

	GATTTGTATT	1

	ATTGATAAAG

	GATTTGTATT	1

	ATTGATAAAG

	GATTTGTATT	1

	ATTGATAAAG

	GATTTGTATT	3

	ATTGATAAAG

	GATTTGTATT	1

	ATTGATAAAG

	GATTTGTATT	2

	ATTGATAAAG

	GATTTGTATT	2

	ATTGATAAAG

	GATTTGTATT	5 (*)

	ATTGATAAAG

	GATTTGTATT	3

	ATTGATAAAG

	GATTTGTATT	3

	ATTGATAAAG

	GATTTGTATT	2

	ATTGATAAAG

	GATTTGTATT	1

	ATTGATAAAG

	GATTTGTATT	1

	ATTGATAAAG

	GATTTGTATT	3

	ATTGATAAAG

	GATTTGTATT	1

	ATTGATAAAG

	GATTTGTATT	0

	ATTGATAAAG

[0242]

As can be seen, the maximum homology between the two selected subsequences is 50 percent (5 matches out of the total length of 10), and so these two sequences are compatible with each other.
[0243]

A 10mer subsequence can be selected from the third 24mer sequence of Table I, and pairwise compared to each of the first two 10mer sequences to determine its compatability therewith, etc. and in this way a family of 10mer sequences developed.
[0244]

It is within the scope of this invention, to obtain families of sequences containing 11mer, 12mer, 13mer, 14mer, 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer and 23mer sequences by analogy to that shown for 10mer sequences.
[0245]

It may be desirable to have a family of sequences in which there are sequences greater in length than the 24mer sequences shown in Table I. It is within the capability of a person skilled in the art, given the family of sequences shown in Table I, to obtain such a family of sequences. One possible approach would be to insert into each sequence at one or more locations a nucleotide, non natural base or analogue such that the longer sequence should not have greater similarity than any two of the original non cross hybridizing sequences of Table I and the addition of extra bases to the tag sequences should not result in a major change in the thermodynamic properties of the tag sequences of that set for example the GC content must be maintained between 10%-40% with a variance from the average of 20%. This method of inserting bases could be used to obtain a family of sequences up to 40 bases long.
[0246]

Given a particular family of sequences that can be used as a family of tags (or tag complements), e.g., those of Table I or Table II, or the combined sequences of these two tables, a skilled person will readily recognize variant families that work equally as well.
[0247]

Again taking the sequences of Table I for example, every T could be converted to an A and vice versa and no significant change in the cross-hybridization properties would be expected to be observed. This would also be true if every G were converted to a C.
[0248]

Also, all of the sequences of a family could be taken to be constructed in the 5′-3′ direction, as is the convention, or all of the constructions of sequences could be in the opposition direction (3′-5′).
[0249]

There are additional modifications that can be carried out. For example, C has not been used in the family of sequences. Substitution of C in place of one or more T's of a particular sequence would yield a sequence that is at least as low in homology with every other sequence of the family as the particular sequence chosen to be modified was. It is thus possible to substitute C in place of one or more T's in any of the sequences shown in Table I. Analogously, substituting of C in place of one or more A's is possible, or substituting C in place of one or T's is possible.
[0250]

It is preferred that the sequences of a given family are of the same, or roughly the same length. Preferably, all the sequences of a family of sequences of this invention have a length that is within five bases of the base-length of the average of the family. More preferably, all sequences are within four bases of the average base-length. Even more preferably, all or almost all sequences are within three bases of the average base-length of the family. Better still, all or almost all sequences have a length that is within two of the base-length of the average of the family.
[0251]

It is also possible for a person skilled in the art to derive sets of sequences from the family of sequences that is the subject of this patent and remove sequences that would be expected to have undesirable hybridization properties.
[0000]

Methods for Synthesis of Oligonucleotide Families
[0252]

Preferably oligonucleotide sequences of the invention are synthesized directly by standard phosphoramidite synthesis approaches and the like (Caruthers et al, Methods in Enzymology; 154, 287-313: 1987; Lipshutz et al, Nature Genet.; 21, 20-24: 1999; Fodor et al, Science; 251, 763-773: 1991). Alternative chemistries involving non natural bases such as peptide nucleic acids or modified nucleosides that offer advantages in duplex stability may also be used (Hacia et al; Nucleic Acids Res; 27: 4034-4039, 1999; Nguyen et al, Nucleic Acids Res.; 27, 1492-1498: 1999; Weiler et al, Nucleic Acids Res.; 25, 2792-2799:1997). It is also possible to synthesize the oligonucleotide sequences of this invention with alternate nucleotide backbones such as phosphorothioate or phosphoroamidate nucleotides. Methods involving synthesis through the addition of blocks of sequence in a step wise manner may also be employed (Lyttle et al, Biotechniques, 19: 274-280 (1995). Synthesis may be carried out directly on the substrate to be used as a solid phase support for the application or the oligonucleotide can be cleaved from the support for use in solution or coupling to a second support.
[0000]

Solid Phase Supports
[0253]

There are several different solid phase supports that can be used with the invention. They include but are not limited to slides, plates, chips, membranes, beads, microparticles and the like. The solid phase supports can also vary in the materials that they are composed of including plastic, glass, silicon, nylon, polystyrene, silica gel, latex and the like. The surface of the support is coated with the complementary sequence of the same.
[0254]

In preferred embodiments, the family of tag complement sequences are derivatized to allow binding to a solid support. Many methods of derivatizing a nucleic acid for binding to a solid support are known in the art (Hermanson G., Bioconjugate Techniques; Acad. Press: 1996). The sequence tag may be bound to a solid support through covalent or non-covalent bonds (Iannone et al, Cytometry; 39: 131-140, 2000; Matson et al, Anal. Biochem.; 224: 110-106, 1995; Proudnikov et al, Anal Biochem; 259: 34-41, 1998; Zammatteo et al, Analytical Biochemistry; 280:143-150, 2000). The sequence tag can be conveniently derivatized for binding to a solid support by incorporating modified nucleic acids in the terminal 5′ or 3′ locations.
[0255]

A variety of moieties useful for binding to a solid support (e.g., biotin, antibodies, and the like), and methods for attaching them to nucleic acids, are known in the art. For example, an amine-modified nucleic acid base (available from, eg., Glen Research) may be attached to a solid support (for example, Covalink-NH, a polystyrene surface grafted with secondary amino groups, available from Nunc) through a bifunctional crosslinker (e.g., bis(sulfosuccinimidyl suberate), available from Pierce). Additional spacing moieties can be added to reduce steric hindrance between the capture moiety and the surface of the solid support.
[0000]

Attaching Tags to Analytes for Sorting
[0256]

A family of oligoucleotide tag sequences can be conjugated to a population of analytes most preferably polynucleotide sequences in several different ways including but not limited to direct chemical synthesis, chemical coupling, ligation, amplification, and the like. Sequence tags that have been synthesized with primer sequences can be used for enzymatic extension of the primer on the target for example in PCR amplification.
[0000]

Detection of Single Nucleotide Polymorphisms Using Primer Extension
[0257]

There are a number of areas of genetic analysis where families of non cross hybridizing sequences can be applied including disease dagnosis, single nucleotide polymorphism analysis, genotyping, expression analysis and the like. One such approach for genetic analysis referred to as the primer extension method (also known as Genetic Bit Analysis (Nikiforov et al, Nucleic Acids Res.; 22, 4167-4175: 1994; Head et al Nucleic Acids Res.; 25, 5065-5071: 1997)) is an extremely accurate method for identification of the nucleotide located at a specific polymorphic site within genomic DNA. In standard primer extension reactions, a portion of genomic DNA containing a defined polymorphic site is amplified by PCR using primers that flank the polymorphic site. In order to identify which nucleotide is present at the polymorphic site, a third primer is synthesized such that the polymorphic position is located immediately 3′ to the primer. A primer extension reaction is set up containing the amplified DNA, the primer for extension, up to 4 dideoxynucleoside triphosphates, each labelled with a different fluorescent dye and a DNA polymerase such as the Klenow subunit of DNA Polymerase 1. The use of dideoxy nucleotides ensure that a single base is added to the 3′ end of the primer, a site corresponding to the polymorphic site. In this way the identity of the nucleotide present at a specific polymorphic site can be determined by the identity of the fluorescent dye-labelled nucleotide that is incorporated in each reaction. One major drawback to this approach is its low throughput. Each primer extension reaction is carried out independently in a separate tube.
[0258]

Universal sequences can be used to enhance the throughput of primer extension assay as follows. A region of genomic DNA containing multiple polymorphic sites is amplified by PCR. Alternately, several genomic regions containing one or more polymorphic sites each are amplified together in a multiplexed PCR reaction. The primer extension reaction is carried out as described above except that the primers used are chimeric, each containing a unique universal tag at the 5′ end and the sequence for extension at the 3′ end. In this way, each gene-specific sequence would be associated with a specific universal sequence. The chimeric primers would be hybridized to the amplified DNA and primer extension carried out as described above. This would result in a mixed pool of extended primers, each with a specific fluorescent dye characteristic of the incorporated nucleotide. Following the primer extension reaction, the mixed extension reactions are hybridized to an array containing probes that are reverse complements of the universal sequences on the primers. This would segregate the products of a number of primer extension reactions into discrete spots. The fluorescent dye present at each spot would then identify the nucleotide incorporated at each specific location.
[0000]

Kits Using Families of Tag Sequences
[0259]

The families of non cross-hybridizing sequences may be provided in kits for use in for example genetic analysis. Such kits include at least one set of non cross hybridizing sequences in solution or on a solid support. Preferably the sequences are attached to microparticles and are provided with buffers and reagents that are appropriate for the application. Reagents may include enzymes, nucleotides, fluorescent labels and the like that would be required for specific applications. Instructions for correct use of the kit for a given application will be provided.

EXAMPLES

Example 1

[0000]

Demonstrate Non Cross Talk Behavior
[0260]

One hundred oligonucleotide probes corresponding to a family of non-cross talking oligonucleotides from Table I were synthesized by Integrated DNA Technologies (IDT, Coralville Iowa). These oligonucleotides incorporated a C₆aminolink group coupled to the 5′ end of the oligo through a C₁₈ethylene glycol spacer. These probes were used to prepare microarrays as follows. The probes were resuspended at a concentration of 50 μM in 150 mM NaPO4, pH 8.5. The probes were spotted onto the surface of a SuperAldehyde slide (Telechem Int., Sunnyvale Calif.) using and SDDC-II microarray spotter (ESI, Toronto Ont). The spots formed were approximately 120 FM in diameter with 200 μM centre-to-centre spacing. Each probe was spotted 8 times on each microarray. Following spotting, the arrays were processed essentially as described by the slide manufacturer. Briefly, the arrays were treated with 67 mM sodium borohydride in PBS/EtOH (3:1) for 5 minutes then washed with 4 changes of 0.1% SDS. The arrays were not boiled.
[0261]

One hundred labelled oligonucleotide targets were also synthesized by IDT. The sequence of these targets corresponded to the reverse complement of the 100 probe sequences. The targets were labelled at the 5′ end with Cy3.
[0262]

Each Cy3-labeled target oligonucleotide was hybridized separately to two microarrays each of which contained all 100 oligonucleotide probes. Hybridizations were carried out at 42° C. for 2 hours in a 40 ll reaction and contained 40 nM of the labelled target suspended in 10 mM Tris HCl, pH 8.3, 50 mM KCl, 0.1% Tween 20. These are low stringency hybridization conditions designed to provide a rigorous test of the performance of the family of non-cross hybridizing sequences. Hybridizations were carried out by depositing the hybridization solution on a clean cover slip then carefully positioning the microarray slide over the cover slip in order to avoid bubbles. The slide was then inverted and transferred to a humid chamber for incubation. Following hybridization, the cover slip was removed and the microarray was washed in hybridization buffer for 15 minutes at room temperature. The slide was then dried by brief centrifugation.
[0263]

Hybridized microarrays were scanned using a ScanArray Lite (GSI-Lumonics, Billerica Mass.). The laser power and photomultiplier tube voltage used for scanning each hybridized microarray were optimized in order to maximize the signal intensity from the spots representing the perfect match.
[0264]

The results of a sample hybridization are shown in FIG. 1. FIG. 1 a shows the hybridization pattern seen when a microarray containing all 100 probes was hybridized with the target complementary to probe 181234. The 4 sets of paired spots correspond to the probe complementary to the target. FIG. 1 b shows the pattern seen when a similar array was hybridized with a mix of all 100 targets.

Example 2

Tag Sequences Used in Sorting Polynucleotides

[0265]

The family of non cross hybridizing sequence tags or a subset thereof can be attached to oligonucleotide probe sequences during synthesis and used to generate amplified probe sequences. In order to test the feasibility of PCR amplification with non cross hybridizing sequence tags and subsequently addressing each respective sequence to its appropriate location on two-dimensional or bead arrays, the following experiment was devised. A 24mer tag sequence was connected in a 5′-3′ specific manner to a p53 exon specific sequence (20mer reverse primer). The connecting p53 sequence represented the inverse complement of the nucleotide gene sequence. To facilitate the subsequent generation of single stranded DNA post-amplification the tag-Reverse primer was synthesized with a phosphate modification (PO₄) on the 5′-end. A second PCR primer was also generated for each desired exon, which represented the Forward (5′-3′) amplification primer. In this instance the Forward primer was labeled with a 5′-biotin modification to allow detection with Cy3-avidin or equivalent.

[0266]

A practical example of the aforementioned description is as follows: For exon 1 of the human p53 tumor suppressor gene sequence the following tag-Reverse primer was generated:


222087 222063

5′-P04-GATTGTAAGATTTGATAAAGTGTA-TCCAGGGAAGCGTGTCACCGTCGT-3′

Tag Sequence # 3 Exon 1 Reverse

[0267]

The numbering above the Exon-1 reverse primer represents the genomic nucleotide positions of the indicated bases. The corresponding Exon-1 Forward primer sequence is as follows:

221873 221896

5′-Biotin-TCATGGCGACTGTCCAGCTTTGTG-3′
[0268]

In combination these primers will amplify a product of 214 bp plus a 24 bp tag extension yielding a total size of 0.238 bp. Once amplified, the PCR product was purified using a QIAquick PCR purification kit and the resulting DNA was quantified. To generate single stranded DNA the DNA was subjected to—exonuclease digestion thereby resulting in the exposure of a single stranded sequence (anti-tag) complementary to the tag-sequence covalently attached to the solid phase array. The resulting product was heated to 95° C. for 5 minutes and then directly applied to the array at a concentration of 10-50 nM. Following hybridization and concurrent sorting, the tag-Exon 1 sequences were visualized using Cy3-streptavidin. In addition to direct visualization of the biotinylated product, the product itself can now act as a substrate for further analysis of the amplified region, such as SNP detection and haplotype determination.
[0269]

The present invention also includes a family of 1168 24mer polynucleotides that have been demonstrated to be minimally cross-hybridizing with each other. This family of polynucleotides is thus useful as a family of tags, and their complements as tag complements.
[0270]

In order to be considered for inclusion into the family, a sequence had to satisfy a certain number of rules regarding its composition. For example, repetitive regions that present potential hybridization problems such as four or more of a similar base (e.g., AAAA or TTTT) or pairs of Gs were forbidden. Another rule is that each sequence contains exactly six Gs and no Cs, in order to have sequences that are more or less isothermal. Also required for a 24mer to be included is that there must be at most six bases between every neighboring pair of Gs. Another way of putting this is that there are at most six non-Gs between any two consecutive Gs. Also, each G nearest the 5′-end (resp. 3′-end) of its oligonucleotide (the left-hand (resp. right-hand) side as written in Table II) was required to occupy one of the first to seventh positions (counting the 5′-terminal (resp. 3′-terminal) position as the first position.)
[0271]

The process used to design families of sequences that do not exhibit cross-hybridization behavior is illustrated generally in FIG. 5). Depending on the application for which these families of sequences will be used, various rules are designed. A certain number of rules can specify constraints for sequence composition (such as the ones described in the previous paragraph). The other rules are used to judge whether two sequences are too similar. Based on these rules, a computer program can derive families of sequences that exhibit minimal or no cross-hybridization behavior. The exact method used by the computer program is not crucial since various computer programs can derive similar families based on these rules. Such a program is for example described in international patent application No. PCT/CA 01/00141 published under WO 01/59151 on Aug. 16, 2001. Other, programs can use different methods, such as the ones summarized below.
[0272]

A first method of generating a maximum number of minimally cross-hybridizing polynucleotide sequences starts with any number of non-cross-hybridizing sequences, for example just one sequence, and increases the family as follows. A certain number of sequences is generated and compared to the sequences already in the family. The generated sequences that exhibit too much similarity with sequences already in the family are dropped. Among the “candidate sequences” that remain, one sequence is selected and added to the family. The other candidate sequences are then compared to the selected sequence, and the ones that show too much similarity are-dropped. A new sequence is selected from the remaining candidate sequences, if any, and added to the family, and soon until there are no candidate sequences left. At this stage, the process can be repeated (generating a certain number of sequences and comparing them to the sequences in the family, etc.) as often as desired. The family obtained at the end of this method contains only minimally cross-hybridizing sequences.
[0273]

A second method of generating a maximum number of minimally cross-hybridizing polynucleotide sequences starts with a fixed-size family of polynucleotide sequences. The sequences of this family can be generated randomly or designed by some other method. Many sequences in this family may not be compatible with each other, because they show too much similarity and are not minimally cross-hybridizing. Therefore, some sequences need to be replaced by new ones, with less similarity. One way to achieve this consists of repeatedly replacing a sequence of the family by the best (that is, lowest similarity) sequence among a certain number of (for example, randomly generated) sequences that are not part of the family. This process can be repeated until the family of sequences shows minimal similarity, hence minimal cross-hybridizing, or until a set number of replacements has occurred. If, at the end of the process, some sequences do not obey the similarity rules that have been set, they can be taken out of the family, thus providing a somewhat smaller family that only contains minimally cross-hybridizing sequences. Some additional rules can be added to this method in order to make it more efficient, such as rules to determine which sequence will be replaced.
[0274]

Such methods have been used to obtain the 1168 non-cross-hybridizing tags of Table II that are also the subject of this patent application.
[0275]

One embodiment of the invention is a composition comprising molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on the group of sequences set out in Table IIA, wherein each of the numeric identifiers 1 to 3-(see the Table) is a nucleotide base selected to be different from the others of 1 to 3. According to this embodiment, several different families of specific sets of oligonucleotide sequences are described, depending upon the assignment of bases made to the numeric identifiers 1 to 3.
[0276]

The sequences contained in Table II have a mathematical relationship to each other, described as follows.

[0277]

Let S and T be two DNA sequences of lengths s and t respectively. While the term “alignment” of nucleotide sequences is widely used in the field of biotechnology, in the context of this invention the term has a specific meaning illustrated here. An alignment of S and T is a 2xp matrix A (with p 2 s and p≧t) such that the first (or second) row of A contains the characters of S (or T respectively) in order, interspersed with p−s (or p−t respectively) spaces. It assumed that no column of the alignment matrix contains two spaces, i.e., that any alignment in which a column contains two spaces is ignored and not considered here. The columns containing the same base in both rows are called matches, while the columns containing different bases are called mismatches. Each column of an alignment containing a space in its first row is called an insertion and each column containing a space in its second row is called a deletion while a column of the alignment containing a space in either row is called an indel. Insertions and deletions within a sequence are represented by the character ‘-’. A gap is a continuous sequence of spaces in one of the rows (that is neither immediately preceded nor immediately followed by another space in the same row), and the length of a gap is the number of spaces in that gap. An internal gap is one in which its first space is preceded by a base and its last space is followed by a base and an internal indel is an belonging to an internal gap. Finally, a block is a continuous sequence of matches (that is neither immediately preceded nor immediately followed by another match), and the length of a block is the number of matches in that block. In order to illustrate these definitions, two sequences S=TGATCGTAGCTACGCCGCG (of length s=19; SEQ ID NO:1169) and T=CGTACGATTGCAACGT (of length t=16, SEQ ID NO:1170) are considered. Exemplary alignment R₁of S and T (with p=23) is:



Alignment R₁:


—	—	—	—	T	G	A	T	C	G	T	A	G	C	T	A	C	G	C	C	G	C	G
C	G	T	A	C	G	A	T	—	—	T	—	G	C	A	A	C	G	T	—	—	—	—
1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23

[0278]

Columns 1 to 4, 9, 10, 12 and 20 to 23 are indels, columns 6, 7, 8, 11, 13, 14, 16, 17 and 18 are matches, and columns 5, 15 and 19 are mismatches. Columns 9 and 10 form a gap of length 2, while columns 16 to 18 form a block of length 3. Columns 9, 10 and 12 are internal indels.
[0279]
A score is assigned to the alignment A of two sequences by assigning weights to each of matches, mismatches and gaps as follows:
- - the reward for a match m,
  - the penalty for a mismatch mm,
  - the penalty for opening a gap og
  - the penalty for extending a gap eg.
    Once these values are set, a score to each column of the alignment is assigned according to the following rules:
  - 1. assign 0 to each column preceding the first match and to each column following the last match.
  - 2. for each of the remaining columns, assign m if it is a match, mm if it is a mismatch, -og-eg if it is the first indel of a gap, -eg if it is an indel but not the first indel of a gap.
    The score of the alignment A is the sum of the scores of its columns. An alignment is said to be of maximum score if no other alignment of the same two sequences has a higher score (with the same values of m, mm, og and eg). A person knowledgeable in the field will recognize this method of scoring an alignment as scoring a local (as opposed to global) alignment with affine gap penalties (that is, gap penalties that can distinguish between the first indel of a gap and the other indels). It will be appreciated that the total number of indels that open a gap is the same as the total number of gaps and that an internal indel is not one of those assigned a 0 in rule (1) above. It will also be noted that foregoing rule (1) assigns a 0 for non-internal mismatches. An internal mismatch is a mismatch that is preceded and followed (not necessarily immediately) by a match.

[0286]

As an illustration, if the values of m, mm, og and eg are set to 3, 1, 2 and 1 respectively, alignment R₁has a score of. 19, determined as shown below:



Scoring of Alignment R₁


—	—	—	—	T	G	A	T	C	G	T	A	G	C	T	A	C	G	C	C	G	C	G
C	G	T	A	C	G	A	T	—	—	T	—	G	C	A	A	C	G	T	—	—	—	—
0	0	0	0	0	3	3	3	−3	−1	3	−3	3	3	−1	3	3	3	0	0	0	0	0

Note that for two given sequences S and T, there are numerous alignments. There are often several alignments of maximum score.

[0287]
Based on these alignments, five sequence similarity measures are defined as follows. For two sequences S and T, and weights {m, mm, og, eg}:
- - M1 is the maximum number of matches over all alignments free of internal indels;
  - M2 is the maximum length of a block over all alignments;
  - M3 is the maximum number of matches over all alignments of maximum score;
  - M4 is the maximum sum of the lengths of the longest two blocks over all alignments of maximum score;
  - M5 is the maximum sum of the lengths of all the blocks of length at least 3, over all alignments of maximum score.
    Notice that, by definition, the following inequalities between these similarity measures are obtained: M4≦M3 and M5≦M3. Also, in order to determine M2 it is sufficient to determine the maximum length of a block over all alignments free of internal indels. For two given sequences, the values of M3 to M5 can vary depending on the values of the weights {m, mm, og, eg}, but not M1 and M2.
[0293]
For weights {3, 1, 2, 1}, the illustrated alignment is not a maximum score alignment of the two example sequences. But for weights {6, 6, 0, 6} it is; hence this alignment shows that for these two example sequences, and weights {6, 6, 0, 6}, M2≧3, M3≧9, M4≧6 and M5≧6. In order to determine the exact values of M1 to M5, all the necessary alignments need to be considered. M1 add M2 can be found by looking at the s+t−1 alignments free of internal indels, where s and t are the lengths of the two sequences considered. Mathematical tools known as dynamic programming can be implemented on a computer and used to determine M3 to M5 in a very quick way. Using a computer program to do these calculations, it was determined that:
- - with the weights {6, 6, 0, 6}, M1=8, M2=4, M3=10, M4=6 and M5=6;
  - with the weights {3, 1, 2, 1}, M1=8, M2=4, M3=10, M4=6 and M5=4.
    According to the preferred embodiment of this invention, two sequences S and T each of length 24 are too similar if at least one of the following happens:
  - M1>16 or
  - M2>13 or
  - M3>20 or
  - M4>16 or
  - M5>19
    when using either weights {6, 6, 0, 6}, or {6, 6, 5, 1}, or {6, 2, 5, 1}, or {6, 6, 6, 0}. In other words, the five similarity measures between S and T are determined for each of the above four sets of weights, and checked against these thresholds (for a total of 20 tests).
[0301]

The above thresholds of 16, 13, 20, 16 and 19, and the above sets of weights, were used to obtain the sequences listed in Table I. Additional sequences can thus be added to those of Table I as long as the above alignment rules are obeyed for all sequences.
[0302]
It is also possible to alter thresholds M1, M2, etc., while remaining within the scope of this invention. It is thus possible to substitute or add sequences to those of Table II, or more generally to those of Table IIA to obtain other sets of sequences that would also exhibit reasonably low cross-hybridization. More specifically, a set of 24mer sequences in which there are no two sequences that are too similar, where too similar is defined as:
- - M1>19 or
  - M2>17 or
  - M3>21 or
  - M4>18 or
  - M5>20
    when using either weights {6, 6, 0, 6}, or {6, 6, 5, 1}, or {6, 2, 5, 1}, or {6, 6, 6, 0}, would also exhibit low cross-hybridization. Reducing any of the threshold values provides sets of sequences with even lower cross-hybridization. Alternatively, ‘too similar’ can also be defined as:
  - M1>19 or
  - M2>17 or
  - M3>21 or
  - M4>18 or
  - M5>20
    when using either weights {3, 1, 2, 1}. Alternatively, other combinations of weights will lead to sets of sequences with low cross-hybridization.
[0313]

Notice that using weights {6, 6, 0, 6} is equivalent to using weights {1, 1, 0, 1}, or weights {2, 2, 0, 2}, . . . (that is, for any two sequences, the values of M1 to M5 are exactly the same whether weights {6, 6, 0, 6} or {1, 1, 0, 1} or {2, 2, 0, 2} or any other multiple of {1, 1, 0, 1} is used).
[0314]
When dealing with sequences of length other than 24, or sequences of various lengths, the definition of similarity can be adjusted. Such adjustments are obvious to the persons skilled in the art. For example, when comparing a sequence of length L1 with a sequence of length L2 (with L1<L2), they can be considered as too similar when
- M1>19/24×L1
- M2>17/24×L1
- M3>21/24×L1
- M4>18/24×L1
- M5>20/24×L1
  when using either weights {6, 6, 0, 6}, or {6, 6, 5, 1}, or {6, 2, 5, 1} or {6, 6, 6, 0}.
[0320]

Polynucleotide sequences can be composed of a subset of natural bases most preferably A, T and G. Sequences that are deficient in one base possess useful characteristics, for example, in reducing potential secondary structure formation or reduced potential for cross hybridization with nucleic acids in nature. Also, it is preferable to have tag sequences that behave isothermally. This can be achieved for example by maintaining a constant base composition for all sequences such as six Gs and eighteen As or Ts' for each sequence. Additional sets of sequences can be designed by extrapolating on the original family of non-cross-hybridizing sequences by simple methods known to those skilled in the art.
[0321]

In order to validate the sequence set, a subset of sequences from the family of 1168 sequence tags was selected and characterized, in terms of the ability of these sequences to form specific duplex structures with their complementary sequences, and the potential for cross-hybridization within the sequence set. See Example 4, below. The subset of 100 sequences was randomly selected, and analyzed using the Luminex¹⁰⁰LabMAP™ platform. The 100 sequences were chemically immobilized onto the set of 100 different Luminex microsphere populations, such that each specific sequence was coupled to one spectrally distinct microsphere population. The pool of 100 microsphere-immobilized probes was then hybridized with each of the 100 corresponding complementary sequences. Each sequence was examined individually for its specific hybridization with its complementary sequence, as well as for its non-specific hybridization with the other 99 sequences present in the reaction. This analysis demonstrated the propensity of each sequence to hybridize only to its complement (perfect match), and not to cross-hybridize appreciably with any of the other oligonucleotides present in the hybridization reaction.
[0322]

It is within the capability of a person skilled in the art, given the family of sequences of Table II, to modify the sequences, or add other sequences while largely retaining the property of minimal cross-hybridization which the polynucleotides of Table II have been demonstrated to have.
[0323]

There are 1168 polynucleotide sequences given in Table II. Since all 1168 of this family of polynucleotides can work with each other as a minimally cross-hybridizing set, then any plurality of polynucleotides that is a subset of the 1168 can also act as a minimally cross-hybridizing set of polynucleotides. An application in which, for example, 30 molecules are to be sorted using a family of polynucleotide tags and tag complements could thus use any group of 30 sequences shown in Table II. This is not to say that some subsets may be found in a practical sense to be more preferred than others. For example, it may be found that a particular subset is more tolerant of a wider variety of conditions under which hybridization is conducted before the degree of cross-hybridization becomes unacceptable.
[0324]

It may be desirable to use polynucleotides that are shorter in length than the 24 bases of those in Table II. A family of subsequences (i.e., subframes of the sequences illustrated) based on those contained in Table II having as few as 10 bases per sequence could be chosen, so long as the subsequences are chosen to retain homological properties between any two of the sequences of the family important to their non cross-hybridization.
[0325]

The selection of sequences using this approach would be amenable to a computerized process. Thus for example, a string of 10 contiguous bases of the first 24mer of Table II could be selected: AAATTGTGAAAGATTGTTTGTGTA (SEQ ID NO:1).
[0326]

The same string of contiguous bases from the second 24mer could then be selected and compared for similarity against the first chosen sequence: GTTAGAGTTAATTGTATTTGATGA (SEQ ID NO:2 of Table II). A systematic pairwise comparison could then be carried out to determine if the similarity requirements are violated. If the pair of sequences does not violate any set property, a 10mer subsequence can be selected from the third 24mer sequence of Table II, and compared to each of the first two 10mer sequences (in a pairwise fashion to determine its compatibility therewith, etc. In this way a family of 10mer sequences may be developed.
[0327]

It is within the scope of this invention, to obtain families of sequences containing 1mer, 12mer, 13mer, 14mer, 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer and 23mer sequences by analogy to that shown for 10mer sequences. It may be desirable to have a family of sequences in which there are sequences greater in length than the 24mer sequences shown in Table II. It is within the capability of a person skilled in the art, given the family of sequences shown in Table II, to obtain such a family of sequences. One possible approach would be to insert into each sequence at one or more locations a nucleotide, non-natural base or analogue such that the longer sequence should not have greater similarity than any two of the original non-cross-hybridizing sequences of Table II and the addition of extra bases to the tag sequences should not result in a major change in the thermodynamic properties of the tag sequences of that set for example the GC content must be maintained between 10%-40% with a variance from the average of 20%. This method of inserting bases could be used to obtain, for example, a family of sequences up to 40 bases long.
[0328]

Given a particular family of sequences that can be used as a family of tags (or tag complements), e.g., those of Table II, a skilled person will readily recognize variant families that work equally as well.
[0329]

Again taking the sequences of Table II for example, every T could be converted to an A and vice versa and no significant change in the cross-hybridization properties would be expected to be observed. This would also be true if every G were converted to a C.
[0330]

Also, all of the sequences of a family could be taken to be constructed in the 5′-3′ direction, as is the convention, or all of the constructions of sequences could be in the opposition direction (3′-5′).
[0331]

There are additional modifications that can be carried out. For example, C has not been used in the family of sequences. Substitution of C in place of one or more G's of a particular sequence would yield a sequence that is at least as low in homology with every other sequence of the family as was the particular sequence chosen for modification. It is thus possible to substitute C in place of one or more G's in any of the sequences shown in Table II. Analogously, substituting of C in place of one or more A's is possible, or substituting C in place of one or T's is possible.
[0332]

It is preferred that the sequences of a given family are of the same, or roughly the same length. Preferably, all the sequences of a family of sequences of this invention have a length that is within five bases of the base-length of the average of the family. More preferably, all sequences are within four bases of the average base-length. Even more preferably, all or almost all sequences are within three bases of the average base-length of the family. Better still, all or almost all sequences have a length that is within two of the base-length of the average of the family, and even better still, within one of the base-length of the average of the family.
[0333]

It is also possible for a person skilled in the art to derive sets of sequences from the family of sequences described in this specification and remove sequences that would be expected to have undesirable hybridization properties.

Example 3

Cross Talk Behavior of Sequence on Beads

[0334]

A group of 100 of the sequences of Table I was tested for feasibility for use as a family of minimally cross-hybridizing oligonucleotides. The 100 sequences selected are separately indicated in Table I along with the numbers assigned to the sequences in the tests.
[0335]

The tests were conducted using the Luminex LabMAP™ platform available from Luminex Corporation, Austin, Tex., U.S.A. The one hundred sequences used as probes; were synthesized as oligonucleotides by Integrated DNA Technologies (IDT, Coralville, Iowa, U.S.A.). Each probe included a C₆aminolink group coupled to the 5′-end of the oligonucleotide through a C₁₂ethylene glycol spacer. The C₆aminolink molecule is a six carbon spacer containing an amine group that can be used for attaching the oligonucleotide to a solid support. One hundred oligonucleotide targets (probe complements), the sequence of each being the reverse complement of the 100 probe sequences, were also synthesized by IDT. Each target was labelled at its 5′-end with biotin. All oligonucleotides were purified using standard desalting procedures, and were reconstituted to a concentration of approximately 200 μM in sterile, distilled water for use. Oligonucleotide concentrations were determined spectrophotometrically using extinction coefficients provided by the supplier.
[0336]

Each probe was coupled by its amino linking group to a carboxylated fluorescent microsphere of the LabMAP system according to the Luminex¹⁰⁰protocol. The microsphere, or bead, for each probe sequence has unique, or spectrally distinct, light absorption characteristics which permits each probe to be distinguished from the other probes. Stock bead pellets were dispersed by sonication and then vortexing. For each bead population, approximately five million microspheres (400 μL) were removed from the stock tube using barrier tips and added to a 1.5 mL Eppendorf tube (USA Scientific). The microspheres were then centrifuged, the supernatant was removed, and beads were resuspended in 25 μL of 0.2 M MES (2-(N-morpholino)ethane sulfonic acid) (Sigma), pH 4.5, followed by vortexing and sonication. One nmol of each probe (in a 25 μL volume) was added to its corresponding bead population. A volume of 2.5 μL of EDC cross-linker (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Pierce), prepared immediately before use by adding 1.0 mL of sterile ddH₂O to 10 mg of EDC powder, was added to each microsphere population. Bead mixes were then incubated for 30 minutes at room temperature in the dark with periodic vortexing. A second 2.5 μL aliquot of freshly prepared EDC solution was then added followed by an additional 30 minute incubation in the dark. Following the second EDC incubation, 1.0 mL of 0.02% Tween-20 (BioShop) was added to each bead mix and vortexed. The microspheres were centrifuged, the supernatant was removed, and the beads were resuspended in 1.0 mL of 0.1% sodium dodecyl sulfate (Sigma). The beads were centrifuged again and the supernatant removed. The coupled beads were resuspended in 100 μL of 0.1 M MES pH 4.5. Bead concentrations were then determined by diluting each preparation 100-fold in ddH₂O and enumerating using a Neubauer BrightLine Hemacytometer. Coupled beads were stored as individual populations at 2-8° C. protected from light.
[0337]

The relative oligonucleotide probe density on each bead population was assessed by Terminal Deoxynucleotidyl Transferase (TdT) end-labelling with biotin-ddUTPs. TdT was used to label the 3′-ends of single-stranded DNA with a labeled ddNTP. Briefly, 180 μL of the pool of 100 bead populations (equivalent to about 4000 of each bead type) to be used for hybridizations was pipetted into an Eppendorf tube and centrifuged. The supernatant was removed, and the beads were washed in 1×TdT buffer. The beads were then incubated with a labelling reaction mixture, which consisted of 5×TdT buffer, 25 mM CoCl₂, and 1000 pmol of biotin-16-ddUTP (all reagents were purchased from Roche). The total reaction volume was brought up to 85.5 μL with sterile, distilled H₂O, and the samples were incubated in the dark for 1 hour at 37° C. A second aliquot of enzyme was added, followed by a second 1 hour incubation. Samples were run in duplicate, as was the negative control, which contained all components except the TdT. In order to remove unincorporated biotin-ddUTP, the beads were washed 3 times with 200 μL of hybridization buffer, and the beads were resuspended in 50 μL of hybridization buffer following the final wash. The biotin label was detected spectrophotometrically using SA-PE (streptavidin-phycoerythrin conjugate). The streptavidin binds to biotin and the phycoerythrin is spectrally distinct from the probe beads. The 10 mg/mL stock of SA-PE was diluted 100-fold in hybridization buffer, and 15 μL of the diluted SA-PE was added directly to each reaction and incubated for 15 minutes at 37° Celsius. The reactions were analyzed on the Luminex¹⁰⁰LabMAP. Acquisition parameters were set to measure 100 events per bead using a sample volume of 50 μL.
[0338]

The results obtained are shown in FIG. 2. As can be seen the Mean Fluorescent Intensity (MFI) of the beads varies from 277.75 to 2291.08, a range of 8.25-fold. Assuming that the labelling reactions are complete for all of the oligonucleotides, this illustrates the signal intensity that would be obtained for each type of bead at this concentration if the target (i.e., labelled complement) was bound to the probe sequence to the full extent possible.
[0339]

The cross-hybridization of targets to probes was evaluated as follows. 100 oligonucleotide probes linked to 100 different bead populations, as described above, were combined to generate a master bead mix, enabling multiplexed reactions to be carried out. The pool of microsphere-immobilized probes was then hybridized individually with each biotinylated target. Thus, each target was examined individually for its specific hybridization with its complementary bead-immobilized sequence, as well as for its non-specific hybridization with the other 99 bead-immobilized universal sequences present in the reaction. For each hybridization reaction, 25 μL bead mix (containing about 2500 of each bead population in hybridization buffer) was added to each well of a 96-well Thermowell PCR plate and equilibrated at 37° C. Each target was diluted to a final concentration of 0.002 fmol/μL in hybridization buffer, and 25 μL (50 fmol) was added to each well, giving a final reaction volume of 50 μL. Hybridization buffer consisted of 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 and hybridizations were performed at 37° C. for 30 minutes. Each target was analyzed in triplicate and six background samples (i.e. no target) were included in each plate. A SA-PE conjugate was used as a reporter, as described above. The 10 mg/mL stock of SA-PE was diluted 100-fold in hybridization buffer, and 15 μL of the diluted SA-PE was added directly to each reaction, without removal of unbound target, and incubated for 15 minutes at 37° C. Finally, an additional 35 μL of hybridization buffer was added to each well, resulting in a final volume of 100 μL per well prior to analysis on the Luminex¹⁰⁰LabMAP. Acquisition parameters were set to measure 100 events per bead using a sample volume of 80 μL.
[0340]

The percent hybridization was calculated for any event in which the NET MFI was at least 3 times the zero target background. In other words, a calculation was made for any sample where (MFI_sample−MFI_{zero target background})/MFI_{zero target background}≧3.
[0341]

A “positive” cross-talk event (i.e., significant mismatch or cross-hybridization) was defined as any event in which the net median fluorescent intensity (MFI_sample−MFI_{zero target background}) generated by a mismatched hybrid was greater than or equal to the arbitrarily set limit of 10% that of the perfectly matched hybrid determined under identical conditions. As there are 100 probes and 100 targets, there are 100×100=10,0000 possible different interactions possible of which 100 are the result of perfect hybridizations. The remaining 9900 result from hybridization of a target with a mismatched probe.
[0342]

The results obtained are illustrated in FIG. 3. The ability of each target to be specifically recognized by its matching probe is shown of the possible 9900 non-specific hybridization events that could have occurred when the 100 targets were each exposed to the pool of 100 probes, 6 events were observed. Of these 6 events, the highest non-specific event generated a signal equivalent to 10.2% of the signal observed for the perfectly matched pair (i.e. specific hybridization event).
[0343]

Each of the 100 targets was thus examined individually for specific hybridization with its complement sequence as incorporated onto a microsphere, as well as for non-specific hybridization with the complements of the other 99 target sequences. Representative hybridization results for target 16 (complement of probe -16, Table I) are shown in FIG. 4. Probe 16 was found to hybridize only to its perfectly-matched target. No cross-hybridization with any of the other 99 targets was observed.
[0344]

The foregoing results demonstrate the possibility of incorporating the 210 sequences of Table I, or any subset thereof, into a multiplexed system with the expectation that most if not all sequences can be distinguished from the others by hybridization. That is, it is possible to distinguish each target from the other targets by hybridization of the target with its precise complement and minimal hybridization with complements of the other targets.
[0000]

Methods for Synthesis of Oligonucleotide Families
[0345]

Preferably oligonucleotide sequences of the invention are synthesized directly by standard phosphoramidite synthesis approaches and the like (Caruthers et al, Methods in Enzymology; 154, 287-313: 1987; Lipshutz et al, Nature Genet.; 21, 20-24: 1999; Fodor et al, Science; 251, 763-773: 1991). Alternative chemistries involving non natural bases such as peptide nucleic acids or modified nucleosides that offer advantages in duplex stability may also be used (Hacia et al; Nucleic Acids Res; 27: 4034-4039, 1999; Nguyen et al, Nucleic Acids Res.; 27, 1492-1498: 1999; Weiler et al, Nucleic Acids Res.; 25, 2792-2799:1997). It is also possible to synthesize the oligonucleotide sequences of this invention with alternate nucleotide backbones such as phosphorothioate or phosphoroamidate nucleotides. Methods involving synthesis through the addition of blocks of sequence in a stepwise-manner may also be employed (Lyttle et al, Biotechniques, 19: 274-280 (1995). Synthesis may be carried out directly on the substrate to be used as a solid phase support for the application or the oligonucleotide can be cleaved from the support for use in solution or coupling to a second support.
[0000]

Solid Phase Supports
[0346]

There are several different solid phase supports that can be used with the invention. They include but are not limited to slides, plates, chips, membranes, beads, microparticles and the like. The solid phase supports can also vary in the materials that they are composed of including plastic, glass, silicon, nylon, polystyrene, silica gel, latex and the like. The surface of the support is coated with the complementary tag sequences by any conventional means of attachment.
[0347]

In preferred embodiments, the family of tag complement sequences is derivatized to allow binding to a solid support. Many methods of derivatizing a nucleic acid for binding to a solid support are known in the art (Hermanson G., Bioconjugate Techniques; Acad. Press: 1996). The sequence tag may be bound to a solid support through covalent or non-covalent bonds (Iannone et al, Cytometry; 39: 131-140, 2000; Matson et al, Anal. Biochem.; 224: 110-106, 1995; Proudnikov et al, Anal Biochem; 259: 34-41, 1998; Zammatteo et al, Analytical Biochemistry; 280:143-150, 2000). The sequence tag can be conveniently derivatized for binding to a solid support by incorporating modified nucleic acids in the terminal 5′ or 3′ locations.
[0348]

A variety of moieties useful for binding to a solid support (e.g., biotin, antibodies, and the like), and methods for attaching them to nucleic acids, are known in the art. For example, an amine-modified nucleic acid base (available from, eg., Glen Research) may be attached to a solid support (for example, Covalink-NH, a polystyrene surface grafted with secondary amino groups, available from Nunc) through a bifunctional crosslinker (e.g., bis(sulfosuccinimidyl suberate), available from Pierce). Additional spacing moieties can be added to reduce steric hindrance between the capture moiety and the surface of the solid support.
[0000]

Attaching Tags to Analytes for Sorting
[0349]

A family of oligonucleotide tag sequences can be conjugated to a population of analytes most preferably polynucleotide sequences in several different ways including but not limited to direct chemical synthesis, chemical coupling, ligation, amplification, and the like. Sequence tags that have been synthesized with primer sequences can be used for enzymatic extension of the primer on the target for example in PCR amplification.
[0000]

Detection of Single Nucleotide Polymorphisms Using Primer Extension
[0350]

There are a number of areas of genetic analysis where families of non-cross-hybridizing sequences can be applied including disease diagnosis, single nucleotide polymorphism analysis, genotyping, expression analysis and the like. One such approach for genetic analysis, referred to as the primer extension method (also known as Genetic Bit Analysis (Nikiforov et al, Nucleic Acids Res.; 22, 4167-4175: 1994; Head et al Nucleic Acids Res.; 25, 5065-5071: 1997)), is an extremely accurate method for identification of the nucleotide located at a specific polymorphic site within genomic DNA. In standard primer extension reactions, a portion of genomic DNA containing a defined polymorphic site is amplified by PCR using primers that flank the polymorphic site. In order to identify which nucleotide is present at the polymorphic site, a third primer is synthesized such that the polymorphic position is located immediately 3′ to the primer. A primer extension reaction is set up containing the amplified DNA, the primer for extension, up to 4 dideoxynucleoside triphosphates (each labeled with a different fluorescent dye) and a DNA polymerase such as the Klenow subunit of DNA Polymerase 1. The use of dideoxy nucleotides ensures that a single base is added to the 3′ end of the primer, a site corresponding to the polymorphic site. In this way the identity of the nucleotide present at a specific polymorphic site can be determined by the identity of the fluorescent dye-labeled nucleotide that is incorporated in each reaction. One major drawback to this approach is its low throughput. Each primer extension reaction is carried out independently in a separate tube.
[0351]

Universal sequences can be used to enhance the throughput of primer extension assay as follows. A region of genomic DNA containing multiple polymorphic sites is amplified by PCR. Alternatively, several genomic regions containing one or more polymorphic sites each are amplified together in a multiplexed PCR reaction. The primer extension reaction is carried out as described above except that the primers used are chimeric, each containing a unique universal tag at the 5′ end and the sequence for extension at the 3′ end. In this way, each gene-specific sequence would be associated with a specific universal sequence. The chimeric primers would be hybridized to the amplified DNA and primer extension is carried out as described above. This would result in a mixed pool of extended primers, each with a specific fluorescent dye characteristic of the incorporated nucleotide. Following the primer extension reaction, the mixed extension reactions are hybridized to an array containing probes that are reverse complements of the universal sequences on the primers. This would segregate the products of a number of primer extension reactions into discrete spots. The fluorescent dye present at each spot would then identify the nucleotide incorporated at each specific location. A number of additional methods for the detection of single nucleotide polymorphisms, including but not limited to, allele specific polymerase chain reaction (ASPCR), allele specific primer extension (ASPE) and oligonucleotide ligation assay (OLA) can be performed by someone skilled in the art in combination with the tag sequences described herein.
[0000]

Kits Using Families Of Tag Sequences
[0352]

The families of non cross-hybridizing sequences may be provided in kits for use in for example genetic analysis. Such kits include at least one set of non-cross-hybridizing sequences in solution or on a solid support. Preferably the sequences are attached to microparticles and are provided with buffers and reagents that are appropriate for the application. Reagents may include enzymes, nucleotides, fluorescent labels and the like that would be required for specific applications. Instructions for correct use of the kit for a given application will be provided.

EXAMPLES

Example 4

Cross Talk Behavior of Sequence on Beads

[0353]

A group of 100 sequences, randomly selected from Table II, was tested for feasibility for use as a family of minimally cross-hybridizing oligonucleotides. The 100 sequences selected are separately indicated in Table II along with the numbers assigned to the sequences in the tests.
[0354]

The tests were conducted using the Luminex LabMAP™ platform available from Luminex Corporation, Austin, Tex., U.S.A. The one hundred sequences, used as probes, were synthesized as oligonucleotides by Integrated DNA Technologies (IDT, Coralville, Iowa, U.S.A.). Each probe included a C₆aminolink group coupled to the 5′-end of the oligonucleotide through a C₁₂ethylene glycol spacer. The C₆aminolink molecule is a six carbon spacer containing an amine group that can be used for attaching the oligonucleotide to a solid support. One hundred oligonucleotide targets (probe complements), the sequence of each being the reverse complement of the 100 probe sequences, were also synthesized by IDT. Each target was labelled at its 5′-end with biotin. All oligonucleotides were purified using standard desalting procedures, and were reconstituted to a concentration of approximately 200 μM in sterile, distilled water for use. Oligonucleotide concentrations were determined spectrophotometrically using extinction coefficients provided by the supplier.
[0355]

Each probe was coupled by its amino linking group to a carboxylated fluorescent microsphere of the LapMAP system according to the Luminex¹⁰⁰protocol. The microsphere, or bead, for each probe sequence has unique, or spectrally distinct, light absorption characteristics which permits each probe to be distinguished from the other probes. Stock bead pellets were dispersed by sonication and then vortexing. For each bead population, five million microspheres (400 μL) were removed from the stock tube using barrier tips and added to a 1.5 mL Eppendorf tube (USA Scientific). The microspheres were then centrifuged, the supernatant was removed, and beads were resuspended in 25 μL of 0.2 M MES (2-(N-morpholino)ethane sulfonic acid) (Sigma), pH 4.5, followed by vortexing and sonication. One nmol of each probe (in a 25 μL volume) was added to its corresponding bead population. A volume of 2.5 μL of EDC cross-linker (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Pierce), prepared immediately before use by adding 1.0 mL of sterile ddH₂₀to 10 mg of EDC powder, was added to each microsphere population. Bead mixes were then incubated for 30 minutes at room temperature in the dark with periodic vortexing. A second 2.5 μL aliquot of freshly prepared. EDC solution was then added followed by an additional 30 minute incubation in the dark. Following the second EDC incubation, 1.0 mL of 0.02% Tween-20 (BioShop) was added to each bead mix and vortexed. The microspheres were centrifuged, the supernatant was removed, and the beads were resuspended in 1.0 mL of 0.1% sodium dodecyl sulfate (Sigma). The beads were centrifuged again and the supernatant removed. The coupled beads were resuspended in 100 μL of 0.1 M MES pH 4.5. Bead concentrations were then determined by diluting each preparation 100-fold in ddH₂O and enumerating using a Neubauer BrightLine Hemacytometer. Coupled beads were stored as individual populations at 8° C. protected from light.
[0356]

The relative oligonucleotide probe density on each bead population was assessed by Terminal Deoxynucleotidyl Transferase (TdT) end-labelling with biotin-ddUTPs. TdT was used to label the 3′-ends of single-stranded DNA with a labeled ddNTP. Briefly, 180 μL of the pool of 100 bead populations (equivalent to about 4000 of each bead type) to be used for hybridizations was pipetted into an Eppendorf tube and centrifuged. The supernatant was removed, and the beads were washed in 1×TdT buffer. The beads were then incubated with a labelling reaction mixture, which consisted of 5×TdT buffer, 25 mM CoCl₂, and 1000 pmol of biotin-16-ddUTP (all reagents were purchased from Roche). The total reaction volume was brought up to 85.5 μL with sterile, distilled H₂O, and the samples were incubated in the dark for 1 hour at 37° C. A second aliquot of enzyme was added, followed by a second 1 hour incubation. Samples were run in duplicate, as was the negative control, which contained all components except the TdT. In order to remove unincorporated biotin-ddUTP, the beads were washed 3 times with 200 μL of hybridization buffer, and the beads were resuspended in 50 μL of hybridization buffer following the final wash. The biotin label was detected spectrophotometrically using SA-PE (streptavidin-phycoerythrin conjugate). The streptavidin binds to biotin and the phycoerythrin is spectrally distinct from the probe beads. The 10 mg/mL stock of SA-PE was diluted 100-fold in hybridization buffer, and 15 μL of the diluted SA-PE was added directly to each reaction and incubated for 15 minutes at 37° Celsius. The reactions were analyzed on the Luminex¹⁰⁰LabMAP. Acquisition parameters were set to measure 100 events per bead using a sample volume of 50 μL.
[0357]

The results obtained are shown in FIG. 6. As can be seen the Mean Fluorescent Intensity (MFI) of the beads varies from 840.3 to 3834.9, a range of 4.56-fold. Assuming that the labelling reactions are complete for all of the oligonucleotides, this illustrates the signal intensity that would be obtained for each type of bead at this concentration if the target (i.e., labelled complement) was bound to the probe sequence to the full extent possible.
[0358]

The cross-hybridization of targets to probes was evaluated as follows. 100 oligonucleotide probes linked to 100 different bead populations, as described above, were combined to generate a master bead mix, enabling multiplexed reactions to be carried out. The pool of microsphere-immobilized probes was then hybridized individually with each biotinylated target. Thus, each target was examined individually for its specific hybridization with its complementary bead-immobilized sequence, as well as for its non-specific hybridization with the other 99 bead-immobilized universal sequences present in the reaction. For each hybridization reaction, 25 μL bead mix (containing about 2500 of each bead population in hybridization buffer) was added to each well of a 96-well Thermowell PCR plate and equilibrated at 37° C. Each target was diluted to a final concentration of 0.002 fmol/LL in hybridization buffer, and 25 μL (50 fmol) was added to each well, giving a final reaction volume of 50 μL. Hybridization buffer consisted of 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 and hybridizations were performed at 37° C. for 30 minutes. Each target was analyzed in triplicate and six background samples (i.e. no target) were included in each plate. A SA-PE conjugate was used as a reporter, as described above. The 10 mg/mL stock of SA-PE was diluted 100-fold in hybridization buffer, and 15 μL of the diluted SA-PE was added directly to each reaction, without removal of unbound target, and incubated for 15 minutes at 37° C. Finally, an additional 35 μL of hybridization buffer was added to each well, resulting in a final volume of 100 μL per well prior to analysis on the Luminex¹⁰⁰LabMAP. Acquisition parameters were set to measure 100 events per bead using a sample volume of 80 μL.
[0359]

The percent hybridization was calculated for any event in which the NET MFI was at least 3 times the zero target background. In other words, a calculation was made for any sample where (MFI_sample−MFI_{zero target background})/MFI_{zero target background}≧3.
[0360]

The net median fluorescent intensity (MFI_sample−MFI_{zero target background}) generated for all of the 10,000 possible target/probe combinations was calculated. As there are 100 probes and 100 targets, there are 100×100=10,0000 possible different interactions possible of which 100 are the result of perfect hybridizations. The remaining 9900 result from hybridization of a target with a mismatched probe. A cross-hybridization event is then defined as a non-specific event whose net median fluorescent intensity exceeds 3 times the zero target background. In other words, a cross-talk calculation is only be made for any sample where (MFI_sample−MFI_{zero target background})/MFI_{zero target background}≧3. Cross-hybridization events were quantified by expressing the value of the cross-hybridization signal as a percentage of the perfect match hybridization signal with the same probe.
[0361]

The results obtained are illustrated in FIG. 7. The ability of each target to be specifically recognized by its matching probe is shown of the possible 9900 non-specific hybridization events that could have occurred when the 100 targets were each exposed to the pool of 100 probes, 6 events were observed. Of these 6 events, the highest non-specific event generated a signal equivalent to 5.3% of the signal observed for the perfectly matched pair (i.e. specific hybridization event).
[0362]

Each of the 100 targets was thus examined individually for specific hybridization with its complement sequence as incorporated onto a microsphere, as well as for non-specific hybridization with the complements of the other 99 target sequences. Representative hybridization results for target (complement of probe 90, Table II) are shown in FIG. 8. Probe 90 was found to hybridize only to its perfectly-matched target. No cross-hybridization with any of the other 99 targets was observed.
[0363]

The foregoing results demonstrate the possibility of incorporating the 1168 sequences of Table II, or any subset thereof, into a multiplexed system with the expectation that most if not all sequences can be distinguished from the others by hybridization. That is, it is possible to distinguish each target from the other targets by hybridization of the target with its precise complement and minimal hybridization with complements of the other targets.

Example 5

Tag Sequences Used in Sorting Polynucleotides

[0364]

The family of non cross hybridizing sequence tags or a subset thereof can be attached to oligonucleotide probe sequences during synthesis and used to generate amplified probe sequences. In order to test the feasibility of PCR amplification with non cross hybridizing sequence tags and subsequently addressing each respective sequence to its appropriate location on two-dimensional or bead arrays, the following experiment was devised. A 24mer tag sequence can be connected in a 5′-3′ specific manner to a p53 exon specific sequence (20mer reverse primer). The connecting p53 sequence represents the inverse complement of the nucleotide gene sequence. To facilitate the subsequent generation of single stranded DNA post-amplification the tag-Reverse primer can be synthesized with a phosphate modification (PO₄) on the 5′-end. A second PCR primer can also be generated for each desired exon, represented by the Forward (5′-3′) amplification primer. In this instance the Forward primer can be labeled with a 5′-biotin modification to allow detection with Cy3-avidin or equivalent.

[0365]

A practical example of the aforementioned description is as follows: For exon 1 of the human p53 tumor suppressor gene sequence the following tag-Reverse primer (SEQ ID NO: 1171) can be generated:


222087 222063

5′-P04- -TCCAGGGAAGCGTGTCACCGTCGT-3′

Tag Sequence # 3 Exon 1 Reverse

[0366]

The numbering above the Exon-1 reverse primer represents the genomic nucleotide positions of the indicated bases. The corresponding Exon-1 Forward primer sequence (SEQ ID NO:1172) is as follows:

221873 221896

5′-Biotin-TCATGGCGACTGTCCAGCTTTGTG-3′
[0367]

In combination, these primers will amplify a product of 214 bp plus a 24 bp tag extension yielding a total size of 238 bp. Once amplified, the PCR product can be purified using a QIAquick PCR purification kit and the resulting DNA can be quantified. To generate single stranded DNA, the DNA is subjected to λ-exonuclease digestion thereby resulting in the exposure of a single stranded sequence (anti-tag) complementary to the tag-sequence covalently attached to the solid phase array. The resulting product is heated to 95° C. for 5 minutes and then directly applied to the array at a concentration of 10-50 nM. Following hybridization and concurrent sorting, the tag-Exon 1 sequences are visualized using Cy3-streptavidin. In addition to direct visualization of the biotinylated product, the product itself can now act as a substrate for further analysis of the amplified region, such as SNP detection and haplotype determination.
[0368]

The Invader Assay is described in detail in U.S. Pat. Nos. 5,846,717 and 5,985,557. Briefly, the ability of the Invader technology to identify target nucleic acid sequences and in particular single base pair changes is dependent on the proper structure being formed, followed by subsequent recognition and cleavage of this structure by the Cleavase enzyme. For recognition by Cleavase III, the target sequence must be complementary to the primary probe, and there must be at least a 1 base “invasion” (overlap) of this structure by an upstream oligonucleotide. Cleavable “flaps’ can be created by invasion of an upstream oligonucleotide without primer extension, and the site of cleavage is determined by the extent to which the upstream oligonucleotide overlaps the 5′ region of the downstream oligonucleotide. Cleavage by the Cleavase enzyme is dependent on this invaded structure and is sensitive to single-base mismatches is positioned immediately upstream of the cleavage site. By adding overlapping pairs of oligonucleotide probes complementary to a predetermined region of target DNA, the cleavage of the downstream probes become a sensitive indicator of the presence of the target sequence. Further, reaction conditions have been established that allow multiple copies of the downstream oligonucleotide probe to be cleaved for each target sequence without temperature cycling, so as to amplify the cleavage signal and allow quantitative detection of target DNA at sub-attomole levels. Incorporation of the minimally cross-hybridizing sequences of the invention described herein into the probe that will be cleaved by the Cleavase enzyme allows detection of multiple target DNA sequences in a single experiment.
[0000]

Definitions
[0369]

Non-cross-hybridization: Describes the absence of hybridization between two sequences that are not perfect complements of each other.
[0370]

Cross-hybridization: The hydrogen bonding of a single-stranded DNA sequence that is partially but not entirely complementary to a single-stranded substrate.
[0371]

Homology or Similarity: How closely related two or more separate strands of DNA are to each other, based on their base sequences.
[0372]

Analogue: The symbols A, G, T/U, C take on their usual meaning in the art here. In the case of T and U, a person skilled in the art would understand that these are equivalent to each other with respect to the inter-strand hydrogen-bond (Watson-Crick) binding properties at work in the context of this invention. The two bases are thus interchangeable and hence the designation of T/U. A chemical, which resembles a nucleotide base is an analogue thereof. A base that does not normally appear in DNA but can substitute for the ones, which do, despite minor differences in structure. Analogues particularly useful in this invention are of the naturally occurring bases can be inserted in their respective places where desired. Such an analogue is any non-natural base, such as peptide nucleic acids and the like that undergoes normal Watson-Crick pairing in the same way as the naturally occurring nucleotide base to which it corresponds.
[0373]

Complement: The opposite or “mirror” image of a DNA sequence. A complementary DNA sequence has an “A” for every “T” and a “C” for every “G”. Two complementary strands of single stranded DNA, for example a tag sequence and its complement, will join to form a double-stranded molecule.
[0374]

Complementary DNA (cDNA): DNA that is synthesized from a messenger RNA template; the single-stranded form is often used as a probe in physical mapping.
[0375]

Oligonucleotide: Refers to a short nucleotide polymer whereby the nucleotides may be natural nucleotide bases or analogues thereof.
[0376]

Tag: Refers to an oligonucleotide that can be used for specifically sorting analytes with at least one other oligonucleotide that when used together do not cross hybridize.
[0377]

Similar Homology: In the context of this invention, pairs of sequences are compared with each other based on the amount of “homology” between the sequences. By way of example, two sequences are said to have a 50% “maximum homology” with each other if, when the two sequences are aligned side-by-side with each other so to obtain the (absolute) maximum number of identically paired bases, the number of identically paired bases is 50% of the total number of bases in one of the sequences. (If the sequences being compared are of different lengths, then it would be of the total number of bases in the shorter of the two sequences.) Examples of determining maximum homology are as follows:

Example 1

[0378]


* *

A-A-B-B-C-C

B-D-C-D-D-D	(2 out of 4 paired bases are the
	same)

* *

A-A-B-B-C-C

B-D-C-D-D-D	(2 out of 3 paired bases are the
	same)

[0379]

In this case, the maximum number of identically paired bases is two and there are two possible alignments yielding this maximum number. The total number of possible pairings is six giving 33⅓% ( 2/6) homology. The maximum amount of homology between the two sequences is thus ⅓.

Example 2

[0380]


* * *

A-A-B-B-C-A

A-A-D-D-C-D	(3 out of 6 paired bases are the same)

[0381]

In this alignment, the number of identically paired bases is three and the total number of possibly paired bases is six, so the homology between the two sequences is 3/6(50%).

*

A-A-B-B-C-A

A-A-D-D-C-D (1 out of 1 paired bases are

the same)
[0382]

In this alignment, the number of identically paired bases is 1, so the homology between the two sequences is ⅙ (16⅔%)
[0383]

The maximum homology between these two sequences is thus 50%.
[0384]

Block sequence: Refers to a symbolic representation of a sequence of blocks. In its most general form a block sequence is a representative sequence in which no particular value., mathematical variable, or other designation is assigned to each block of the sequence.
[0385]

Incidence Matrix: As used herein is a well-defined term in the field of Discrete Mathematics. However, an incidence matrix cannot be defined without first defining a “graph”. In the method described herein a subset of general graphs called simple graphs is used. Members of this subcategory are further defined as follows.
[0386]

A simple graph G is a pair (V, E) where V represents the set of vertices of the simple graph and E is a set of un-oriented edges of the simple graph. An edge is defined as a 2-component combination of members of the set of vertices. In other words, in a simple graph G there are some pairs of vertices that are connected by an edge. In our application a graph is based on nucleic acid sequences generated using sequence templates and vertices represent DNA sequences and edges represent a relative property of any pair of sequences.
[0387]
The incidence matrix is a mathematical object that allows one to describe any given graph. For the subset of simple graphs used herein, the simple graph G=(V,E), and for a pre-selected and fixed ordering of vertices, V={v₁,v₂, . . . v_n}, elements of the incidence matrix A(G)=[a_ij] are defined by the following rules:
- - (1) a_ij=1 for any pair of vertices {v_i,v_j} that is a member of the set of edges; and
  - (2) a_ij=0 for any pair of vertices {v_i,v_j}that is not a member of the set of edges.
    This is an exact unequivocal definition of the incidence matrix. In effect, one selects the indices: 1, 2, . . . n of the vertices and then forms an (n×n) square matrix with elements a_ij=1 if the vertices v_iand v_jare connected by an edge and a_ij=0 if the vertices v_iand v_jare not connected by an edge.
[0390]

To define the term “class property” as used herein, the term “complete simple graph” or “clique” must first be defined. The complete simple graph is required because all sequences that result from the method described herein should collectively share the relative property of any pair of sequences defining an edge of graph G, for example not violating the threshold rule that is, do not have a “maximum simple homology” greater than a predetermined amount, whatever pair of the sequences are chosen from the final set. It is possible that additional “local” rules, based on known or empirically determined behavior of particular nucleotides, or nucleotide sequences, are applied to sequence pairs in addition to the basic threshold rule.
[0391]

In the language of a simple graph, G=(V, E), this means in the final graph there should be no pair of vertices (no sequence pair) not connected by an edge (because an edge means that the sequences represented by v_iand v_jdo not violate the threshold rule).
[0392]

Because the incidence matrix of any simple graph can be generated by the above definition of its elements, the consequence of defining a simple complete graph is that the corresponding incidence matrix for a simple complete graph will have all off-diagonal elements equal to 1 and all diagonal elements equal to 0. This is because if one aligns a sequence with itself, the threshold rule is of course violated, and all other sequences are connected by an edge.
[0393]

For any simple graph, there might be a complete subgraph. First, the definition of a subgraph of a graph is as follows. The subgraph Gs=(Vs, Es) of a simple graph G=(V, E) is a simple graph that contains the subsets of vertices Vs of the set V of vertices and inclusion of the set Vs into the set V is immersion (a mathematical term). This means that one generates a subgraph Gs=(Vs,Es) of a simple graph G in two steps. First select some vertices Vs from G. Then select those edges Es from G that connect the chosen vertices and do not select edges that connect selected with non selected vertices.
[0394]

We desire a subgraph of G that is a complete simple graph. By using this property of the complete simple graph generated from the simple graph G of all sequences generated by the template based algorithm, the pairwise property of any pair of the sequences (violating/non-violating the threshold rule) is converted into the property of all members of the set, termed “the class property”.
[0395]

By selecting a subgraph of a simple graph G that is a complete simple graph, this assures that, up to the tests involving the local rules described herein, there are no pairs of sequences in the resulting set that violate the threshold rule, also described above, independent of which pair of sequences in the set are chosen. This feature is called the “desired class property”.

[0396]

The present invention thus includes reducing the potential for non cross-hybridization behavior by taking into account local homologies of the sequences and appears to have greater rigor than known approaches. For example, the method described herein involves the sliding of one sequence relative to the other sequence in order to form a sequence alignment that would accommodate insertions or deletions. (Kane et al., Nucleic Acids Res.; 28, 4552-4557: 2000).

TABLE I


		No. Assigned
SEQ ID NO (1)	Sequence	in Example 3


1	GATTTGTATTGATTGAGATTAAAG	1

2	TGATTGTAGTATGTATTGATAAAG	2

3	GATTGTAAGATTTGATAAAGTGTA	3

4	GATTTGAAGATTATTGGTAATGTA	4

5	GATTGATTATTGTGATTTGAATTG	5

6	GATTTGATTGTAAAAGATTGTTGA	6

7	ATTGGTAAATTGGTAAATGAATTG	7

8	ATTGGATTTGATAAAGGTAAATGA

9	GTAAGTAATGAATGTAAAAGGATT	8

10	GATTGATTGATTGATTGATTTGAT

11	TGATGATTAAAGAAAGTGATTGAT

12	AAAGGATTTGATTGATAAAGTGAT

13	TGTAGATTTGTATGTATGTATGAT	10

14	GATTTGATAAAGAAAGGATTGATT

15	GATTAAAGTGATTGATGATTTGTA	11

16	AAAGAAAGAAAGAAAGAAAGTGTA	12

17	TGTAAAAGGATTGATTTGTATGTA

18	AAAGTGTAGATTGATTAAAGAAAG

19	AAAGTTGATTGATTGAAAAGGTAT

20	TTGATTGAGATTGATTTTGAGTAT

21	TGAATTGATGAATGAATGAAGTAT	15

22	GTAATGAAGTATGTATGTAAGTAA

23	TGATGATTTGAATGAAGATTGATT	16

24	TGATAAAGTGATAAAGGATTAAAG	17

25	TGATTTGAGTATTTGAGATTTTGA	18

26	TGTAGTAAGATTGATTAAAGGTAA

27	GTATAAAGGATTGATTTTGAAAAG

28	GTATTTGAGTAAGTAATTGATTGA	19

29	GTAAAAAGTTGAGTATTGAAAAAG

30	GATTTGATAAAGGATTTGTATTGA

31	GATTGTATTGAAGTATTGTAAAAG	20

32	TGATGATTTTGATGAAAAAGTTGA

33	TGATTTGAGATTAAAGAAAGGATT	21

34	TGATTGAATTGAGTAAAAAGGATT	22

35	AAAGTGTAAAAGGATTTGATGTAT

36	AAAGGTATTTGAGATTTGATTGAA

37	AAAGTTGAGATTTGAATGATTGAA	23

38	TGTATTGAAAAGGTATGATTTGAA

39	GTATTGTATTGAAAAGGTAATTGA	24

40	TTGAGTAATGATAAAGTGAAGATT

41	TGAAGATTTGAAGTAATTGAAAAG	25

42	TGAAAAAGTGTAGATTTTGAGTAA	26

43	TGTATGAATGAAGATTTGATTGTA

44	AAAGTTGAGTATTGATTTGAAAAG	27

45	GATTTGTAGATTTGTATTGAGATT

46	AAAGAAAGGATTTGTAGTAAGATT	29

47	GTAAAAAGAAAGGTATAAAGGTAA	30

48	GATTAAAGTTGATTGAAAAGTGAA	31

49	TGAAAAAGGTAATTGATGTATGAA

50	AAAGGATTAAAGTGAAGTAATTGA	33

51	ATGAATTGGTATGTATATGAATGA	34

52	TGAAATGAATGAATGATGAAATTG	35

53	ATTGATTGTGAATGAAATGAATTG	36

54	ATTGAAAGATGAAAAGATGAAAAG	37

55	ATTGTTGAAAAGTGTAATGATTGA	38

56	ATGATGTAATGAAAAGATTGTGTA	39

57	AAAGATTGAAAGATGATGTAATTG

58	ATTGATGAGTATATTGTGTAGTAA	41

59	AAAGATTGTGTAATTGATGATGAA

60	AAAGGTATATTGTGTAATGAGTAA

61	TGTAATGAGTATTGTAATTGAAAG	43

62	GTATAAAGAAAGATTGGTAAATGA	44

63	TTGAGTAATTGAATTGTGAAATGA	45

64	TGTATTGAATGAATTGTTGATGTA	46

65	TGTAATTGGTAAATGAGTAAAAAG

66	TGAATGAAATTGATGAGTATAAAG

67	GTAAGTAAATTGAAAGATTGATGA	49

68	GTAAATGATGATATTGGTATATTG	50

69	ATTGTTGATGATTGATTGAAATGA	51

70	ATTGTGAAGTATAAAGATGATTGA	52

71	ATGAAAAGTTGAGTAAATTGTGAT

72	ATGAATTGAAAGTGATTGAAAAAG	54

73	GTAAATTGATGAAAAGTTGATGAT

74	AAAGTGATGTATATGAGTAAATTG	56

75	GTAATGATAAAGATGATGATATTG	57

76	TTGAAAAGATTGGTAATGATATGA

77	AAAGTGAAAAAGATTGATTGATGA	59

78	ATTGATGAGATTGATTATTGTGTA

79	ATGAGATTATTGGATTTGTAGATT	60

80	TGAAGATTATGAATTGGTAAGATT	61

81	ATTGGATTATGAGATTATGATTGA	62

82	ATTGTTGAATTGGATTAAAGATGA

83	AAAGATGAGTAAGTAAATTGGATT

84	AAAGGTAAGATTATTGATGAAAAG	65

85	ATTGATGAGATTAAAGTTGAATTG

86	GATTATTGGATTATGAAAAGGATT

87	GATTTGTAATTGTTGAGTAAATGA	67

88	AAAGAAAGATTGTTGAGATTATGA	68

89	GTATAAAGGATTTTGAATTGATGA

90	TTGAGATTGTAAATGAATTGTTGA

91	GTATATTGATTGTGTAATGAAAAG

92	TGATATGAATTGGATTATTGGTAT	70

93	ATGAATGATGAATGATGATTATTG

94	ATGAATTGATTGGATTGTAATGAT	71

95	GATTGTAATTGAGTAAATTGATGA

96	GATTATTGGATTAAAGGTAAATGA	72

97	ATTGTTGAATTGATGAGATTTGAT	73

98	GATTATGAGTAAATTGATTGTGAT

99	GATTATTGTTGATGAATGATATTG

100	TGTAAAAGATTGAAAGGTATGATT	75

101	GTATTTAGATGAGTTTGTTAGATT	76

102	TGAAGTTATGTAATAGAAAGTGAT

103	GTATGTATTGTATGTAGTTAATTG	77

104	TGATATAGATAGTTAGATAGATAG	78

105	ATGATGATGTATTGTAGTTATGAA	79

106	TTAGTGAATGTATTAGTTGATGTA

107	GTTAGTTAGATTATTGTTAGTTAG	80

108	GTTAATTGTGTAGTTTGTTATTGA

109	GTTATGAAATAGTGATATTGTTAG

110	ATTGTTAGAAAGTGTAGATTAAAG	81

111	ATGAGTATGTTATTAGTGTATGTA	82

112	TGTAATAGTGAAGTTAGATTGTAT	83

113	ATTGATAGATGATTAGTTAGTTGA	84

114	ATGAGTTTGTTTATGAGATTAAAG

115	TGATGTTTGATTATGATGTAGTAT	85

116	ATGAGTTAGTTATGAATTAGATGA

117	ATTGTTAGTGATGTTAGTAATTAG	86

118	TGATGTAAGTATTGATGTTAGTTT	87

119	GATTGTAAATAGAAAGTGAAGTAA	88

120	ATTGTGTATGAAGTATTGTATGAT

121	ATAGTGATGTTATGAAGATTGTTA

122	TTAGATGAATTGTGAAGTATTTAG	90

123	GTAAGTTATGATTGATGTTATGAA	91

124	GTATTGATGTTTAAAGTGTAATAG	92

125	GATTGTAAGTAAGATTGTATATTG

126	GTTTGTATTTAGATGAATAGAAAG	93

127	GTTTGATTTGTAATAGTGATTGTA

128	TGTATGTAGTATTTAGAAAGATGA

129	ATGAATTGTGATAAAGAAAGTTAG

130	TTAGTGTAGTAAGTTTAAAGTGTA	95

131	GTATGATTGTTTGTAATTAGTGAT

132	GTTTAAAGTTAGTTGAGTTAGTAT	96

133	ATAGTGTATGTAGATTATGAGATT	97

134	TTGAATGATTAGTTGAGTATGATT	98

135	GTATGTAAGTTAGTATGATTTGAA

136	TGTAGTATATTGTTGAATTGTGAT

137	ATAGTGATTGTATGTATGATAAAG

138	TTAGTGATTGATGTATATTGAAAG

139	GTAAGATTATGAGTTATGATGTAA

140	GTTATGAAATTGTTAGTGTAGATT	99

141	GTTAGATTTGTAGTTTAAAGATAG	100

142	TTAGTGATTGAAATGATGTAGATT

143	AAAGTGTAGTTATTAGTTAGTTAG

144	AAAGAAAGTGTATGATGTTATTAG

145	GATTGTATATTGTGTATGATGATT

146	TTGAGATTGTTATGATATGAGTAT

147	ATGAGTATGATTGTTATGATGTTT

148	TGATTTAGTGAAATTGTGTATTAG

149	TGAATGTATGTAGTATGTTTGTTA

150	GTTAGTATTGATGATTATGAGTTA

151	GTATATTGTGATTTAGTTGAGATT

152	GTTAGTTTAAAGTTGAGATTGTTT

153	GTATATTGTTAGATGAGATTTGTA

154	TGATGTATGTTAGTTTATGAATGA

155	TGTAGTATGTAATGTAGTATTTGA

156	ATGAGTTATGTATTGAGTTAGTAT

157	TGTATGATGATTATAGTTGAGTAA

158	ATTGATGAATGAGTTTGTATAAAG

159	TTGAGTTTATGATTAGAAAGAAAG

160	TGATATTGATGAGTTAGTATTGAA

161	ATAGAAAGTGAAATGAGTATGTTA

162	TTGATGTAGATTTGATGTATATAG

163	TTGAGATTATAGTGTAGTTTATAG

164	TGATGTTAGATTGTTTGATTATTG

165	TGTATTAGATAGTGATTTGAATGA

166	GATTATGATGAATGTAGTATGTAA

167	TGAATGATTGATATGAATAGTGTA

168	GTAATGATTTAGTGTATTGAGTTT

169	TGTAGTAATGATTTGATGATAAAG

170	TGAAGATTGTTATTAGTGATATTG

171	GTATTTGAATGATGTAATAGTGTA

172	GTATATGATGTATTAGATTGAAAG

173	AAAGTTAGATTGAAAGTGATAAAG

174	GTAAGATGTTGATATAGAAGATTA	9

175	TAATATGAGATGAAAGTGAATTAG

176	TTAGTGAAGAAGTATAGTTTATTG	13

177	GTAGTTGAGAAGATAGTAATTAAT

178	ATGAGATGATATTTGAGAAGTAAT

179	GATGTGAAGAAGATGAATATATAT

180	AAAGTATAGTAAGATGTATAGTAG	14

181	GAAGTAATATGAGTAGTTGAATAT

182	TTGATAATGTTTGTTTGTTTGTAG	28

183	TGAAGAAGAAAGTATAATGATGAA

184	GTAGATTAGTTTGAAGTGAATAAT	32

185	TATAGTAGTGAAGATGATATATGA

186	TATAATGAGTTGTTAGATATGTTG

187	GTTGTGAAATTAGATGTGAAATAT

188	TAATGTTGTGAATAATGTAGAAAG	40

189	GTTTATAGTGAAATATGAAGATAG	42

190	ATTATGAAGTAAGTTAATGAGAAG	47

191	GATGAAAGTAATGTTTATTGTGAA

192	ATTATTGAGATGTGAAGTTTGTTT	48

193	TGTAGAAGATGAGATGTATAATTA	53

194	TAATTTGAGTTGTGTATATAGTAG

195	TGATATTAGTAAGAAGTTGAATAG

196	GTTAGTTATTGAGAAGTGTATATA	55

197	GTAGTAATGTTAATGAATTAGTAG	58

198	GTTTGTTTGATGTGATTGAATAAT

199	GTAAGTAGTAATTTGAATATGTAG	64

200	GTTTGAAGATATGTTTGAAGTATA

201	ATGATAATTGAAGATGTAATGTTG

202	GTAGATAGTATAGTTGTAATGTTA	66

203	GATGTGAATGTAATATGTTTATAG	69

204	TGAAATTAGTTTGTAAGATGTGTA	74

205	TGTAGTATAAAGTATATGAAGTAG	63

206	ATATGTTGTTGAGTTGATAGTATA	89

207	ATTATTGAGTAGAAAGATAGAAAG	94

208	GTTGTTGAATATTGAATATAGTTG

209	ATGAGAAGTTAGTAATGTAAATAG

210	TGAAATGAGAAGATTAATGAGTTT

[0397]

TABLE II


		No. in
Sequence	SEQ ID NO:	Ex 4


AAATTGTGAAAGATTGTTTGTGTA	1	1

GTTAGAGTTAATTGTATTTGATGA	2	—

ATGTTAAAGTAAGTGTTGAAATGT	3	—

TGATGTTAGAAGTATATTGTGAAT	4	—

TTTGTGTAGAATATGTGTTGTTAA	5	—

ATAAGTGTAAGTGAAATAAGAAGA	6	—

AAGAGTATTTGTTGTGAGTTAAAT	7	—

GTGTTTATGTTATATGTGAAGTTT	8	—

AAAGAGAATAGAATATGTGTAAGT	9	—

TATGAAAGAGTGAGATAATGTTTA	10	—

ATGAGAAATATGTTAGAATGTGAT	11	—

TTAGTTGTTGATGTTTAGTAGTTT	12	—

GTAAAGAGTATAAGTTTGATGATA	13	—

AAAGTAAGAATGATGTAATAAGTG	14	—

GTAGAAATAGTTTATTGATGATTG	15	—

TGTAAGTGAAATAGTGAGTTATTT	16	2

AAATAGATGATATAAGTGAGAATG	17	—

ATAAGTTATAAGTGTTATGTGAGT	18	—

TATAGATAAAGAGATGATTTGTTG	19	—

AGAGTTGAGAATGTATAGTATTAT	20	—

AAGTAGTTTGTAAGAATGATTGTA	21	—

TTATGAAATTGAGTGAAGATTGAT	22	—

GTATATGTAAATTGTTATGTTGAG	23	—

GAATTGTATAAAGTATTAGATGTG	24	4

TAGATGAGATTAAGTGTTATTTGA	25	—

GTTAAGTTTGTTTATGTATAGAAG	26	—

GAGTATTAGTAAAGTGATATGATA	27	—

GTGAATGATTTAGTAAATGATTGA	28	—

GATTGAAGTTATAGAAATGATTAG	29	—

AGTGATAAATGTTAGTTGAATTGT	30	—

TATATAGTAAATGTTTGTGTGTTG	31	—

TTAAGTGTTAGTTATTTGTTGTAG	32	—

GTAGTAATATGAAGTGAGAATATA	33	—

TAGTGTATAGAATGTAGATTTAGT	34	—

TTGTAGATTAGATGTGTTTGTAAA	35	—

TAGTATAGAGTAGAGATGATATTT	36	—

ATTGTGAAAGAAAGAGAAGAAATT	37	7

TGTGAGAATTAAGATTAAGAATGT	38	—

ATATTAGTTAAGAAAGAAGAGTTG	39	—

TTGTAGTTGAGAAATATGTAGTTT	40	—

TAGAGTTGTTAAAGAGTGTAAATA	41	—

GTTATGATGTGTATAAGTAATATG	42	—

TTTGTTAGAATGAGAAGATTTATG	43	10

AGTATAGTTTAAAGAAGTAGTAGA	44	—

GTGAGATATAGATTTAGAAAGTAA	45	—

TTGTTTATAGTGAAGTGAATAGTA	46	—

AAGTAAGTAGTAATAGTGTGTTAA	47	—

ATTTGTGAGTTATGAAAGATAAGA	48	—

GAAAGTAGAGAATAAAGATAAGAA	49	—

ATTTAAGATTGTTAAGAGTAGAAG	50	—

GTTTAAAGATTGTAAGAATGTGTA	51	—

TTTGTGAAGATGAAGTATTTGTAT	52	—

TGTGTTTAGAATTTAGTATGTGTA	53	—

GATAATGATTATAGAAAGTGTTTG	54	—

GTTATTTGTAAGTTAAGATAGTAG	55	—

AGTTTATTGAAAGAGTTTGAATAG	56	—

TTGTGTTTATTGTGTAGTTTAAAG	57	—

ATTGTGAGAAGATATGAAAGTTAT	58	—

TGAGAATGTAAAGAATGTTTATTG	59	13

ATGTGAAAGTTATGATGTTAATTG	60	—

GTTTAGTATTAGTTGTTAAGATTG	61	—

GATTGATATTTGAATGTTTGTTTG	62	14

TGAATTGAAAGTGTAATGTTGTAT	63	—

GATTGTATTGTTGAGAATAGAATA	64	—

AAATTTGAGATTTGTGATAGAGTA	65	—

GTAATTAGATTTGTTTGTTGTTGT	66	—

GTTTGTATTGTTAGTGAATATAGT	67	—

ATGTAGTAGTAGATGTTTATGAAT	68	—

TGTTTAAAGATGATTGAAGAAATG	69	—

TGTGATAATGATGTTATTTGTGTA	70	—

ATAGTTGTGAGAATTTGTAATTAG	71	—

ATAGATGTAAGAGAAATTGTGAAA	72	—

AGATTAAGAGAAGTTAATAGAGTA	73	—

GAAGTAAATTGTGAATGAAAGAAA	74	—

AATGTAAGAAAGAAGATTGTTGTA	75	—

TTTGATTTATGTGTTATGTTGAGT	76	—

GTATTGAGAAATTTGAAGAATGAA	77	—

GAATTGTATGAAATGAATTGTAAG	78	—

TATTGTAGAAGTAAAGTTAGAAGT	79	—

TTTATGTAATGATAAGTGTAGTTG	80	—

ATATAGTTGAAATTGTGATAGTGT	81	—

ATAAGAAATTAGAGAGTTGTAAAG	82	—

GAATTGTGAAATGTGATTGATATA	83	—

AAATAAGTAGTTTAATGAGAGAAG	84	—

GATTAAAGAAGTAAGTGAATGTTT	85	—

TATGTGTGTTGTTTAGTGTTATTA	86	—

GAGTTATATGTAGTTAGAGTTATA	87	—

GAAAGAAAGAAGTGTTAAGTTAAA	88	—

TAGTATTAGTAAGTATGTGATTGT	89	—

TTGTGTGATTGAATATTGTGAAAT	90	—

ATGTGAAAGAGTTAAGTGATTAAA	91	—

GATTGAATGATTGAGATATGTAAA	92	—

AAGATGATAGTTAAGTGTAAGTTA	93	17

TAGTTGTTATTGAGAATTTAGAAG	94	—

TTTATAGTGAATTATGAGTGAAAG	95	—

GATAGATTTAGAATGAATTAAGTG	96	18

TTTGAAGAAGAGATTTGAAATTGA	97	—

ATGAATAAGAGTTGATAAATGTGA	98	—

TGTTTATGTAGTGTAGATTGAATT	99	—

TTTAAGTGAGTTATAGAAGTAGTA	100	19

GATTTATGTGTTTGAAGTTAAGAT	101	—

TAGTTAGAGAAAGTGATAAAGTTA	102	—

GTAATGATAATGAAGTGTATATAG	103	—

AATGAAGTGTTAGTATAGATAGTA	104	—

TAAATTGAGTTTGTTTGATTGTAG	105	—

TAATGAAGAATAAGTATGAGTGTT	106	—

AAATGTAATAGTGTTGTTAGTTAG	107	—

AGAGTTAGTGAAATGTTGTTAAAT	108	—

GAAATAGAAATGTATTGTTTGTGA	109	—

AGTTATAAGTTTGTGAGAATTAAG	110	—

GAGTTTATAGTTAGAATATGTTGT	111	—

AGAGTTATTAGAAGAAGATTTAAG	112	—

GAGTTAATGAAATAAGTATTTGTG	113	—

ATGATGAATAGTTGAAGTATATAG	114	—

ATAGATATGAGATGAAAGTTAGTA	115	—

TATGTAAAGAAAGTGAAAGAAGAA	116	—

TGAATGTAGAAATGAATGTTGAAA	117	—

AATTGAATAGTGTGTGAGTTTAAT	118	—

AGATATTGTTTGATTAATGAAGAG	119	—

AAAGTTGTAAAGTTGAAGATAAAG	120	—

GTTAAGAGATTATGAGATGTATTA	121	—

AGAAGATATAAGAAGATTGAATTG	122	—

GTAGAAATTTGAATTGATGTGAAA	123	—

AAGAGTAGATTGATAAGTATATGA	124	—

TGATATAGTAGTGAAGAAATAAGT	125	22

AGATAATGATGAGAAATGAAGATA	126	—

ATGTGAAAGTATTTGTGATATAGT	127	—

AATAAGAGAATTGATATGAAGATG	128	23

TAAGTGTATTTAGTAGAATGAAGT	129	—

TATGTTAGATTTGTTGAGATTGAT	130	—

AGTTTGTATGAAGAGATAGTATTT	131	—

GAGAAATGTTATGTATTTAGTAGT	132	—

TATGTGAGAATGTGTTTGATTTAA	133	—

GTATGTTTGTTTATAGAATGTATG	134	—

GAGTATATAGAAGAAAGAAATTTG	135	—

ATGAGTGAAGTAAATGTAGTTATT	136	—

TTAAGAAGTGAGTTATTGTGATAT	137	—

ATGAAATGAGAATATTGTTGTTTG	138	—

GATTAATGATTATGTGAATTGATG	139	—

GAAATGTTAAAGATATGAAAGTAG	140	—

TATTGTTGATTTGATATTAGTGTG	141	—

TTTATGTTTGTGTATGTAAGTAGT	142	—

AATTGAAAGAATTGTGTGAATTGA	143	—

TGAGTTTGAATTTGTTTGAGTAAT	144	—

GATGTATAATGATGTGTGTAAATT	145	—

ATGTGAGAGAAGAATTTGTTTATT	146	—

GTGATAAAGTATTGTTGATAGAAA	147	—

GAAGTAGAATAGAAAGTTAATAGA	148	—

TTGTGTAGTTAAGAGTTGTTTAAT	149	24

TAGTAGTAAGTTGTTAGAATAGTT	150	—

AATTTGAAGTATAATGAATGTGTG	151	—

TAGAAATTGTAGTATTTGAGAGAA	152	—

TGTATATGTTAATGAGATGTTGTA	153	25

TATTTGATAAGAGAATGAAGAAGT	154	26

TTGAATAGTGTAATGAATATGATG	155	—

GTAGTTTGTGAATAGAATTAGTTT	156	—

AAAGATGATTGTAATTTGTGTGAA	157	—

GAAGATTGTTGAGTTAATAGATAA	158	—

AGATTATGTAGTGATGTAAATGTT	159	—

GAATTTAGATGTAGATATGAATGT	160	—

GATAGAAGTGTATTAAGTAAGTTA	161	—

TATGAATTATGAGAAGAATAGAGT	162	—

TTTGTTATGAAGTGATTTGTTTGT	163	—

GTAAAGATTGTGTTATATGAAATG	164	—

TTGTGATAGTAGTTAGATATTTGT	165	28

GAATTAAGATAAAGAAGAGAAGTA	166	—

GATTGTAGAATGAATTTGTAGTAT	167	—

AAATAAGAGAGAGAATGATTTAGT	168	—

AATTATGTGAATAGATTGTTGAAG	169	—

TTAAGATTTATGTGATAGTAGAGT	170	—

TTAAAGATAGTGTTTGTTGTGTTA	171	—

TATTGATTTATGAAGAGTATAGTG	172	—

AAATTTGATGAGTAGTTTAAGAGA	173	—

ATAAAGTTGTTTGATGTTTGAATG	174	—

GATTGTGATGAATAATGTTATTGA	175	—

GATGAAGAAATATGATATGAATAG	176	—

TTAAAGTTATTGAAGTGAAGTTGA	177	—

TTGTAAGAAATAGAGATTTGTGTT	178	—

GAGATTGAGTTTAAGTATTAGATT	179	—

AGTGATAATAGAATGATAAATGTG	180	—

GATAATAGTGAATTTGAGTTGTAT	181	—

AGATATTTGTAGTAGAAAGTATGT	182	—

GTTATGAATGTTGAATTTGAATGT	183	—

ATGAAAGATTTAGTTGTGAGATAT	184	30

AAATAGAGAAGTTATGATGTGATA	185	—

TTAGTGAGAAATGTTTAATGTGAT	186	—

TGAAGAATATGTGAAATTAGTTTG	187	—

GTTTGATAGTTTAATGAGTATTGA	188	—

GTTGTAAGTAATGATAAAGTATGA	189	—

TAAGAGTAGTAATTGTTGTTTAGA	190	—

TTTGAGAGAGTATGTATGATTATT	191	—

ATTGATTGTGAATTAGATAGAAGA	192	—

GATTAGTATTTAGTAGTAATAGAG	193	31

TATGTATTAGAGATATTGAAAGTG	194	—

TATGTGAAAGTAATGATAAATGAG	195	—

GTAATTAGTAATGATTTGAATGAG	196	—

GTTTATTGTAAAGATGTAAGTGAA	197	—

TAGTAGAATTGTTGTTAAAGAATG	198	32

TATTGTTAGTTATGTAGTGTGTAA	199	—

GAGTGAAAGTTATATGAAAGTATA	200	—

ATATAGAAGTTGATGAGTTTATGA	201	—

TTTAGAAGTAAGAATAAGTGAGTA	202	—

TGTGTATAAGATATTTGTAAGAAG	203	—

TAGAAGAGTTGTATTGTTATAAGT	204	—

GTGTTATTAGTTTAAGTTAGAGTA	205	—

AATATAGTGATGTGAAATTGAATG	206	—

TTAGAGAATAGAGTGATTATAGTT	207	—

GAAGTGAGTTAATGATTTGTAAAT	208	—

AATGTAAAGTAAAGAAAGTGATGA	209	33

GTTAGTTATGATGAATATTGTGTA	210	34

AAATGAGTTAGAGTAGAATTATGT	211	—

GATATAGAAGATTAGTTAGTGATA	212	—

ATAGTTTGTTGAGATTTATGAGTA	213	—

TAGAATAGTTAGTAGTAAGAGTAT	214	—

GAATTTGTATTGTGAAGTTTAGTA	215	—

GTAGTAAGAAGAGAATTAGATTAA	216	—

AATGTGTTATGTATGTAAATAGTG	217	—

GAATTAGTTAGAGTAAATTGTTTG	218	—

GAAATTGAAGATAGTAAGAAATGA	219	—

GTGTATTATGTGATTTATGATAGA	220	—

TATTATGAGAAAGTTGAATAGTAG	221	35

TATGTATTGTATTGAGTAGATGAA	222	—

GTGATTGAATAGTAGATTGTTTAA	223	36

AGTAAGTTGTTTGATTGAAATTTG	224	—

GAAGTTTGATTTAAGTTTAAGAAG	225	—

GAGAAGATAAATGATATTGTTATG	226	—

ATGATGAGTTGTTAATAGTTAGTT	227	—

TATGATATTTGAAGAGTGTTAAGA	228	—

GAGATGATTAAAGTGATTTATGAA	229	—

ATAGTTAAGAGTGATGAGAATAAA	230	—

TTTATTGTTAGATAAAGAGTTGAG	231	—

AGAATATTGATAGTTGAAGTTGAA	232	—

TAGTGTAAAGTGTAGATTGTAAAT	233	—

AGTAGTGATATGATTTGAATATTG	234	—

TGTATTGAATTAGAATAGTGAGAA	235	—

TGATATGAGATAGAAGTTTAATGT	236	—

GAAGAAGTAAGTATAAAGTAAATG	237	—

TTTAAGTGTGATAAGAAAGATAGA	238	—

TATTGTTGAATGTGTTTAAAGAGA	239	38

GAATAATGATGAGATGATTATTGA	240	—

TAGAGAAAGAGAGAATTGTATTAA	241	39

ATGTATAATGAGATATGTTTGTGA	242	—

AATAGATAAGATTGATTGTGTTTG	243	40

TTTGATGATAATAGAAGAGAATGA	244	—

AGATGAATAAGTTGTGAATGTTTA	245	—

AGATGAAAGAAAGTGTAGAATATT	246	—

TGTTAAATGTATGTAGTAATTGAG	247	41

TAGTAGTGTGAAGTTATTTGTTAT	248	—

AGTGAATGTTTGTAAAGAGTTTAA	249	—

GATAAATGAGAATTGAGTAATTGT	250	—

TGATGAGAAATTGTTTAAGTGTTT	251	—

AAATAAGTAGTGTGAGTAATAGTA	252	—

TATGAAATATGTGATAGTAAGAGA	253	—

ATTGTAAGAGTGATTATAGATGAT	254	—

AGAGTAAGAATGAAAGAGATAATA	255	—

TAAGTAAGTAGATGTTAAAGAGAT	256	—

AAATAGAAAGAATTGTAGAGTAGT	257	—

ATAGATTTAAGTGAAGAGAGTTAT	258	42

GAATGTTTGTAAATGTATAGATAG	259	43

AAATAGAATGAGTAGTGAAATATG	260	—

TTGAATTATGTAGAGAAAGTAAAG	261	—

TAGTAAATTGAGAGTAGTTGAATT	262	—

TGTAAAGTGTTTATAGTGTGTAAT	263	—

ATATGATTTGAGATGAGAATGTAA	264	—

AATATTGATATGTGTTGTGAAGTA	265	—

AGTGAGATTATGAGTATTGATTTA	266	44

TTGTATTTAGATAGTGAGATTATG	267	—

ATAGAAATGAAAGATAGATAGAAG	268	—

GATTGTATATGTAAAGTAGTTTAG	269	—

TATGAATGTTATTGTGTGTTGATT	270	45

GATATTAGTAGAGTAAGTATATTG	271	—

TGAGATGAATTTGTGTTATGATAT	272	—

TATGAATGAAGTAAAGAGATGTAA	273	—

GAGTGAATTTGTTGTAATTTGTTT	274	—

AGAAATTGTAGAGTTAATTGTGTA	275	—

GTGTTAATGAAAGTTGTGAATAAT	276	—

TGTGATTTGTTAAGAAGATTAATG	277	—

AGTAGTATTGTAAAGTATAAAGAG	278	—

TGATTGTTGTATAGTTATTGTGTA	279	—

GATTGTAGTTTAATGTTAAGAATG	280	—

ATGAAATAAGAAATTGAGTAGAGA	281	—

TATGATGATATTTGTTGTATGTGT	282	—

TTTAGAGTTTGATTAGTATGTTTG	283	—

AATAAGAGATTGTGATGAGAAATA	284	—

AATGAATAGAATAGAGAATGTAGA	285	—

GTAGTAGTAATTTGAATGTTTGAA	286	47

AGTGAGTAATTGATTGATTGTTAA	287	—

GAATAATGTTTAGTGTGTTTGAAA	288	—

ATATGAAAGTAGAGAAAGTGTTAT	289	—

TGAGTTATTGTATTTAGTTTGAAG	290	—

TAGTTGAGTTTAAAGTTGAAAGAA	291	—

TAAAGAGTGATGTAAATAGAAGTT	292	—

TGTAGTGTTTAGAGTAAGTTATTA	293	—

AGAGATTAATGTGTTGAAAGATTA	294	—

GTAATAAGTTGTGAAAGAAGATTA	295	—

GAGATGTTATAGATAATGAAAGAA	296	—

TTTAGTTGATTGTTGAATAGAGTA	297	—

ATTATTGAAAGTAGATGTTAGATG	298	—

TTTATGTGTGATTGAGTGTTTAAT	299	—

TATTTAGTTAGATAGATAGAGAGT	300	—

ATGTGTTTATGTGAAAGATTTGTA	301	—

ATAGTAATTAGAAGAGAAGAATGT	302	—

TATGAGTGATTAGAATTGTATTTG	303	—

TTAATGTATTGTTTAAAGAGTGTG	304	—

ATAGAGAATTAAGAATTGTTTGAG	305	—

GTTATAAGTAGAAATGTATAGAAG	306	—

AGTAATTAGTTTGAAATGTGTAGT	307	—

GAAAGATTATGATTGTAAAGTGAT	308	—

GTAAGATTAGAAGTTAATGAAGAA	309	48

GAGAATGTTGAATAAGAAGTAATT	310	—

TTAAGAGTGTTTGAATAGTGTTTA	311	—

ATAAAGAAAGAGTATGAGATTATG	312	—

AGTTATTGATTGAAGATGAGAAAT	313	—

GTTTGTGTTTGTATAAGTTGTTAA	314	50

TTGTATGTGAGTTTAGATTAATGA	315	—

TAGTTAAAGTATAGTTGTTTGAGT	316	—

AAATTTGTGTTGAGATTTGTATAG	317	—

TATTAGTGTTATGATAAAGAGAAG	318	—

TATAAGAAGTAATTTGAGAAGAGT	319	—

TAAGTTGAGATGTTTGTTTGATAA	320	—

GTGTAGATTTATGAATTGAGTAAT	321	—

TATAGAGAAGTGTTTAGTTGTATA	322	—

ATAAAGAAGAATAGTTGTTGTGTA	323	—

AGATTGAAATAGATTAGAAAGTTG	324	—

GTTGTTATAAGAAATAGTTTGTTG	325	—

AGAAATAGAGTAAGAGTGTTTAAA	326	—

AGAGATAGTAGTAAATAGTTATTG	327	—

AAATGATTGTGTAAGTTATGTATG	328	—

AAGAAGTAAGAGAGAAATTTGAAT	329	—

GTGTGTATTTAGTTGATAATTGAT	330	—

ATTGTTGTTGTTGAGAAATGTATT	331	—

AGATAAGTTAAAGTAAAGAGAATG	332	—

TAGTTGAAGTTAGTTTAAGTGTTA	333	—

AGTAAGAATGTAATATGATGATAG	334	—

ATGAGATTGAAAGATTTATGAATG	335	—

TGATTGAATTAGAGAGAATGTATA	336	—

AGTTAGTAAGAGAATATAGTGAAT	337	—

ATTAAGATTGTATAGTTAGTGATG	338	—

GAGATAAAGAATTGAAATAGAAGA	339	—

AGAGTAAATGTTAAGAAAGAAGTT	340	—

AAAGTTTGTTATGTGTGAAGAATT	341	—

ATTGTGTTTAAGAAATATGATGAG	342	—

TATTGAAATGAGATGTATGTAGTT	343	—

ATTTGTGTGATGTTTGAAATATGA	344	—

TAAGATAATAGTGAGAGAAATTGA	345	—

ATTTATGATTAGTGTAAGTGTTGT	346	—

GATTAAGAATAAAGTGTGAAGAAT	347	—

GTAATTGATGAAGAGTTAGTTTAT	348	—

TGTGTTATGTTATAAGAAGTGATA	349	—

AGAGAAATTGAATTTAGAAATGTG	350	—

TTATTGAATGTGAGAAAGTATTTG	351	—

TGTTAATGAGAAGATAATGATAGT	352	—

GAAAGTATTTGTTGATTATTGTTG	353	—

TAGTTTATGTAGTTAATTGTTGAG	354	—

GTTGAAAGATAGTTTGATATGTAT	355	—

TTAGAAGATAGATTATTGAGAAAG	356	—

AATAATGTTGTGAAATAGATGTGA	357	56

AGTAAGAAAGTTTAGTTTAGTTAG	358	—

TAGTTTAATGAGATGTTTGATATG	359	—

TTAAAGATGTTAAAGAATGAGTGA	360	—

AAAGTGTGTATATGTTAGAAAGTA	361	—

ATTAAGTTATGTGTTTATGTGTTG	362	—

TTTGAAGAAGTGTTTGTATTATGT	363	—

TGTTAAGAAGTTTAGTTAAAGTTG	364	—

TTTAAGTATAAGATTGTGTGAGAT	365	—

AGATATTTGATAGATAGAAGAAAG	366	—

ATTTAGAGTTGTAAGAAGATATTG	367	—

GAGAAATTGTAATTGTTAGAGTAT	368	—

GAAGTATATGTTAAGATGTAATAG	369	—

AATATTGAAGATGTAGTGAGTTAT	370	—

GAGTTTAGAAATGATAAAGAATTG	371	—

TAAGAAATGAGTTATATGTTGAGA	372	60

TTGATATAAGAAGTTGTGATAAGT	373	—

AAGTGTTTAATGTAAGAGAATGAA	374	61

GTTGTGAGAATTAGAAATAGTATA	375	—

TTTAGTTTGATGTGTTTATGAGAT	376	—

GTAATTGAAAGTATGAGTAGTAAT	377	—

TAGTTGAATAAGATTGAGAGAAAT	378	—

TTAAGTGAAGTGTTGTTTATTGAA	379	—

ATTGATTTGTTGAAATAAGTGTTG	380	—

TGAATTGTTGATAAGTTATGAAGA	381	—

GTTTGTTATTGAGTAAGTTGAATT	382	—

TGATTTAGTATGTATTAGAGTTGA	383	—

TAAATAGAGATGAGAATAAGAAAG	384	—

AGAATGTTATATGTAGAGAAATTG	385	—

ATTTATGTAGTTGAGAGTGATAAA	386	—

GTAAAGATAGTTTGAGTAATTTGA	387	—

GAAATAGTATAATGTTAAGTGAGA	388	—

ATTGTATATTGTGTTGAAGAAAGT	389	—

GAGTTAAGTGTAAATGAAATGTAA	390	—

ATAGATTGTGTGAAAGAAAGAATT	391	—

TTAATAGAAGTTTGTAGTATGATG	392	—

TTGTATGTGAGAATAAAGTTTAGT	393	—

GTGATTAGATATGATGATATGAAT	394	—

TGAAGAAGAATTTAGATTTGTAAG	395	—

TGTATGATTATTGATTAGTGTGTT	396	—

TGTGAAAGAGAATGATAGATATTT	397	—

AATTGAAATGAGTGTGTTTAAGAA	398	—

ATTATAGAGTTAGTTTAGAATGAG	399	—

AAAGATAGAAATTGAGTGTATGAT	400	—

GTAGTTTGTTAATGTTGTATAATG	401	—

AGAGATATTAGAATGTAAGAATAG	402	64

AGAAGTTTGAAATATGATAGAATG	403	—

TAGAATGTAAAGTTTAGTATAGAG	404	—

AGTAGATGTATGTTAATGTGAATA	405	—

TGAAAGTGAAATATGAAATGTTGT	406	—

ATAGTATATTGAGTTTGTATGAAG	407	—

GAAGAAATGTTTGTAGAATAAGTA	408	—

AATGAGTATTGAAGAAATGTATAG	409	—

GTGATAGAATTTGTGTTTAATGAA	410	66

TGTAGTATGAAGAATAATGAAATG	411	—

ATAGAAGTTAATGATAATTGTGTG	412	—

GTGATTGTAAGTAAGTAAAGATAA	413	—

TATGTAGTTTGTGTTATTTGAAGA	414	—

TGAGTAAGTTTGTATGTTTAAGTA	415	67

TAAATGTATGAGTGTGTAAAGAAA	416	—

GTAAGAGTATTGAAATTAGTAAGA	417	68

GTTGAGTGTAAAGATTATTGATAA	418	—

AGTATGAGTTATTAGATAAAGTGA	419	—

ATTTGTTATAGAGTTGTGTTGTAT	420	—

TAATTAGTAGTGTGTTGAAATTTG	421	—

TGTATTGAGATTGTTATTGTATTG	422	—

GTTATTAGAAGAGATAATTGAGTT	423	—

TTGAGTTGTGATTAAGTAGTATAT	424	—

GATAGTATAATGATTGAAGTAATG	425	—

GTGAAAGATATTTGAGAGATAAAT	426	—

AGTTATGATTTGAAGAAATTGTTG	427	—

GTAAGTATTTGAATTTGATGAGTT	428	—

TAATAGTGTTATAAGTGAAAGAGT	429	—

AAATGAATTGATGTGTATATGAAG	430	—

AGAAAGTGAGTTGTTAAGTATTTA	431	—

TTTATGTGTGAATTGTGTATATAG	432	—

GTAATATGATAGAAATGTAAAGAG	433	—

GAGAATTGTTTAAAGATAGTTGTA	434	—

GAATTTGTTAAGAATGAGTTTGAT	435	—

ATAGTGATGATTAAAGAGAATTTG	436	—

ATAGATGTTTAGTTGAGATTATTG	437	—

AAGAGTGTAAATAGAAAGTGATAT	438	—

TGTGTATTGATTGTTGAGATAAAT	439	—

TAGTATAGTGAGAAAGAGTTAAAT	440	—

AAAGATAAGAAAGAGATGATGTTT	441	—

GAAGTTATTGAAATAGAGAAGTAT	442	—

ATGTATGTATAGAAAGAGTAAATG	443	—

GATGTTTGTAAAGATTGAAATTGA	444	—

AATTTAGAGAGTATTTGTGTTGTA	445	—

AATTTGTTTGAAAGAAAGTAAGTG	446	—

AAAGAGTAGTGTTATTGTTAGATA	447	—

GTATGTTGTATATGTTGTTGATAT	448	—

GTAGAATTTGTTGAGTATTTGTAA	449	—

ATGAATTTAGTTAGTGTAAGAAAG	450	—

ATGATAAGAAATGTTGATGAAGTA	451	—

TTGATGATGAAGATAATGTAGATA	452	—

AGATGATATGATATAGATTAGATG	453	—

TTGAAAGTTAGAAAGATAGATGTT	454	—

GTTTAATGTTAGTTAGAAAGTAAG	455	—

GAGATTTAAGTTTGAAGTGAAATA	456	—

TTTGTTAGTAGTTGTTATAAGAGA	457	—

TATGAGAATAGTTTGTTAGTGAAT	458	—

TTGAAAGTTTAAAGAAGAGATAAG	459	—

AAGTGAGTTGAAATGAAATATGTT	460	—

GTTAGAAATGAAATGAGTAGTTAT	461	—

TAAGTATTGTATTTGTGTGTGTAT	462	—

TGTATTAGTAAAGAAGAGAGAATA	463	—

GAGAAGAGAAATAAGTTGAAATAA	464	—

GTAAAGTAGAAATAGAATTGAGTT	465	—

GTGTGTTATTTGTTTGTAAAGTAT	466	69

TTTGATGTATGAATATAGTATGAG	467	—

AAGATTGTGTGAATAGTTGAAATT	468	—

TATAAAGTTTGAAGATGAGTGATA	469	—

AGATAAAGAGATTTAAGATGTATG	470	71

GAAGAATTAAGTTGAGAATTAAGA	471	—

TAGAGAAATTTGATAAAGAAAGAG	472	—

AAAGTTTATGAAGTTATTGAGTAG	473	—

AAATAGTGTAAGTAAAGAGATGAT	474	—

TATGATGATTTAGTTATAAGAGTG	475	—

TAGATAAATGTTATGATGAGTAAG	476	—

AGATTGATTGTGATGATTTGTATA	477	—

TTAAGAAGAATTGTATATGAGAGT	478	73

GTAGAATGTTTAGAGTTGAATATA	479	—

GAGAAATAGTAAGAAGTAAATAGA	480	—

ATTGAAGTTGTTATGTGAAGATTT	481	—

TAAATGTTGTGTAGAGTAATTAGA	482	—

AAATAAGAGTTTGAGAAGTTGTTT	483	—

AGTTGTAATAAGAAGTGATTTAAG	484	—

GTTAGAATGTATATAGAGTTAGAT	485	74

TTGATATTGAAAGAGAAAGTTATG	486	—

TTAAAGAGAGAAATGTTTGATTAG	487	—

TGTGAATTTGAGTATTAGTAAGAA	488	—

TAATTTGAATGTGAAAGTTGTTAG	489	—

ATGTGTTTGAAAGATGATGATTTA	490	—

AAGTTATGTTGATATTGAGTGAAA	491	—

TAGATAAAGAAGATAGAGATTTAG	492	—

GATGAATGTAGATATATGTAATGA	493	—

GAAGAATAGTTTATGTAAATGATG	494	—

GTAGTATATAGTTAAAGATGAGTT	495	—

GTTATTTGTGTATGATTATGATTG	496	—

AGAGATTAGAAATTGAGAGAATTA	497	—

GTATGATAGAGTTTATAGTGATAA	498	—

GTTAGAAAGAATGAAATTGAAGTA	499	—

AAGAATGAGAATATAGAGATGAAT	500	—

AAAGAGAATAGTGTTTAAGAAGAT	501	—

GATGTGTTATTGATAGAAATTAGA	502	—

TAGAGTTATAGAGATATTGTATGA	503	—

GAGAGTTGAATAAGTTAAAGATAT	504	—

AGATATGAAATAGATTGTTAGAGA	505	—

GAGTGAATAGAAAGATATGTTAAT	506	—

AAAGAGATATTGAAGAGAATAAAG	507	—

GTTATAGAATAAGTTGTAAAGTGT	508	—

TGATAGTATGATAATGTGTTTATG	509	—

TTTGTTGTTAAGTATGTGATTTAG	510	77

TAAAGTGTTGTGTTAAAGATTAAG	511	—

TGTGTTTGATTGATTAATGTTATG	512	—

ATTAATGAATGAGTGTTGTAATGT	513	—

TAGATGTTTGTGAGTTTGATATTA	514	—

GAATGAATAGTAATAGATGATTTG	515	—

AATAGTGTGTTGTTATATGATTAG	516	—

TAGATTAGAAGATGTTGTGTATTA	517	—

AATGTGTGTGTTAAATGAATTTGT	518	—

GAATTAAGTATATGAGTGTAGAAA	519	—

TTATTGTGTGTAAGTAGTGTAAAT	520	—

GTAGTAAAGAGAATTGTTTAGTAT	521	80

AAGTTTGTAAGAAGTAGTTGAATA	522	—

AGTTATAGTATAGTAGTATAGAGA	523	—

GAAAGAAATGTGTATAGTTTAATG	524	—

TTGTGAGTAATGAATGATGTATTA	525	—

GTAGAGTTGTAAATAGAGAATAAA	526	—

ATTAATGTAGATTGTAAGAGATAG	527	—

TTAGTGTGTTTGTAGATAGAATTA	528	—

AGAGAGTTTGTGTATATGTATAAA	529	81

TTAAGTTTAGTGAGATTTGTTAAG	530	—

ATGAAGTTTATTGAATAGTAGTGA	531	—

ATATTTGTGTTGTATGTTTGTGAA	532	—

AAAGTGTTTATAGAAGATTTGATG	533	—

AAGAGATATGATTTGTTAGTTGTA	534	—

AAGAAGAAATGAGTGATAATGTAA	535	—

TAGTGTTTGATATGTTAAGAAGTT	536	—

GTAGAAAGTGATAGATTAGTAATA	537	—

GATAAATGTTAAGTTAGTATGATG	538	—

AGATTAGAAGAATTGTTTAGAATG	539	—

ATATTTGAGAAGTGTGAAATGAAT	540	—

TGAGTAAATAGTTTATGAGTAGTA	541	—

TTAGAGAGTAGATAAAGATTTGAT	542	—

ATTGTTTAAGTTGTTGATAAGATG	543	—

GTTGTAAAGTTAAAGTGTGAATTT	544	—

ATAGATTGTGTGTTTGTTATAGTA	545	—

GTAAGTTATTGAGAATGATAATAG	546	—

TAGATTAGTTGATAAGTGTGTAAT	547	83

AAATGTAAATGAAGAGTGTTTGTT	548	—

GATAGAAGAAATGTATATAGTGAT	549	—

TATAGAGTGTATGTTATGATAAAG	550	—

TATGAAGTGATAAGATGAAGAATT	551	—

TGTTGAGAATAGTAAGAGAATTTA	552	—

TAGATAATGTGAAGTAATAAGTGA	553	84

GTATTATGATGATAGTAGTAAGTA	554	—

AGATATGATTTAGTATTGAATGTG	555	—

AATTAAGTTTGTAGAGTGATTTGA	556	—

AAGAAATAGATGTAGTAAGATGTT	557	—

TTGAGAAGTTGTTGTAATAAGAAT	558	—

AGTGTGAAATAGTGAAAGTTTAAA	559	—

TTTATGTAGTAGATTTATGTGAAG	560	—

ATTAATGAGAAATTAGTGTGTTAG	561	—

ATGTTAATAGTGATAGTAAAGTGA	562	—

TATGTTGATAAATGATTATGAGTG	563	—

TTATTAGAGTTGTGTGTGATATAT	564	—

TGTTGTTATGATTGAGTTAGAATA	565	—

AATTTGAGTTAAGAAGAAGTGTAA	566	—

AAAGATAAAGTTAAGTGTTTGTAG	567	88

TGTTGAGATGATATTGTATAAGTT	568	—

TAAATAGTGAATGAGTTATAGAGT	569	—

ATAGATGTTATGATAGTTAGTTAG	570	—

GTTAAGTGAAGATATGTATTGTTA	571	—

TAAGAAAGTAAAGTTTGTAGATGT	572	—

AAGAGAAAGTTTGATTGAATAAAG	573	—

ATATTAGATGTGAGTTATATGTGT	574	—

AGTTTGAGTTTAGTATTGTGAATA	575	—

ATGTTAAATGAGAGATTGTGTATA	576	—

TAAATGTTGTGATTATTGTGAGAT	577	—

TAAGAATTGAAGTAAGAGTTATTG	578	—

AGAGATAGAATTAAGTTTGTTGAT	579	—

GAAGAATGTTAAGAAATATGTAAG	580	—

TATTTGTGATTAAGAAGTTGAGAA	581	—

AGTTAGAATTTGTGTAGTAGAATT	582	—

AAGTTTATTGTTGATGTTGTATTG	583	—

GAATGAGTTTAAGAGTTTATAGTA	584	—

AGTGAAGATTGTATGTAGTATAAA	585	—

AGTTGAAATGAGTATTAAGTAATG	586	—

ATGTGTTATTTGAGATGAGTAATT	587	—

AAATAGTGTTGTTGAAGTTGTTAT	588	—

GTAGAGAAAGATATATGTAGTTTA	589	—

GAGAGTATTTGATGAATGATTATA	590	—

GAGTATAAGTTTAGTGTATATTGA	591	—

ATAATGTGATTATTGATTGAGAGA	592	—

TTAGTTGTTATGTGAGAGTAATAA	593	—

AAATGAGTATATTGAATTGTGATG	594	—

AATTAGAAGTAAGTAGAGTTTAAG	595	3

TGTAAGTTTAAAGTAAGAAATGTG	596	5

GAAATGATAAGTTGATATAAGAAG	597	—

AATGAGTAGTTTGTATTTGAGTTT	598	—

AGTGAATGTAAGATTATGTATTTG	599	6

GTAATTGAATTGAAAGATAAGTGT	600	8

TATGTTTAAGTAGTGAAATAGAGT	601	—

GTATTGAAATTGAATTAGAAGTAG	602	—

AATATGTAATGTAGTTGAAAGTGA	603	—

TGAATATTGAGAATTATGAGAGTT	604	—

TAGTGTAAATGATGAAGAAAGTAT	605	—

GTATGTGTAAAGAAATTTGATGTA	606	—

AATTGTTTGAAAGTTTGTTGAGAA	607	—

AATTGTTTGAGTAGTATTAGTAGT	608	—

TAATTGAGTTTGAATAAGAGAGTT	609	—

TGTTGATTGTAAGTGTTTATTGTT	610	—

GAAATTTGTGAGTATGTATTTGAA	611	—

TAAGAATGAATGTGAAGTGAATAT	612	—

TAATGTGAAGTTTGTGAAAGATAT	613	—

TTGTATATGAAAGTAAGAAGAAGT	614	—

TAGAGAGAAGAAGAAATAAGAATA	615	—

ATTTGAAATGTTAATGAGAGAGAT	616	—

TTGTGTGTATATAGTATTAGAATG	617	—

ATTGTTAGTATTGATGTGAAGTTA	618	—

TGTTTGTATTTGAATGAAATGAAG	619	—

TGTTAGATTGTGTTAAATGTACTT	620	—

TATAGAGTATTGTATAGACAGAAA	621	—

AAATAGTAAGAATGTACTTGTTGA	622	—

TGAGTGTGATTTATCATTAAGTTA	623	—

ACAATTTGTTGTACTGTTATGATT	624	—

GATTGAAGAAAGAAATAGTTTGAA	625	—

GATAATAGAGAATAGTAGAGTTAA	626	—

GATTGAAATTTGTAGTTATAGTGA	627	—

GATTTAAGAAGATGAATAATGTAG	628	—

TTTGAGAGAAAGTAGAATAAGATA	629	—

GATTAAGAGTAAATGAGTATAAGA	630	—

TTTGATAGAATTGAAATTTGAGAG	631	—

TGAAGAAGAGTGTTATAAGATTTA	632	—

GTGAAATGATTTAGAGTAATAAGT	633	—

AAATAAGAATAGAGAGAGAAAGTT	634	9

GTTGTAAAGTAATAGAGAAATTAG	635	—

AGTGATTTAGATTATGTGATGATT	636	—

AGAGTATAGTTTAGATTTATGTAG	637	—

ATGATTAGATAGTGAAATTGTTAG	638	—

ATGAAATGTATTAGTTTAGAGTTG	639	—

ATATTGAGTGAGAGTTATTGTTAA	640	—

AGATGTGTATTGAATTAAGAAGTT	641	—

TAATGTGTTGATAGAATAGAGATA	642	—

AAATTAGTTGAAAGTATGAGAAAG	643	11

TTTAGAGTTGAAGAAATGTTAATG	644	—

GATTGTTGATTATTGATGAATTTG	645	—

TGTTGTTGTTGAATTGAAGAATTA	646	—

ATTAAGTAAGAATTGAGAGTTTGA	647	12

GTATGTTGTAATGTATTAAGAAAG	648	15

TAGTTGTGATTTATGTAATGATTG	649	—

TGATAATGAAAGTTTATAGAGAGA	650	—

GTAAGATTGTTTGTATGATAAGAT	651	—

TTGAATTAAGAGTAAGATGTTTAG	652	—

AAGTGTTTGTTTAGAGTAAAGATA	653	—

AGAGAGATAAAGTATAGAAGTTAA	654	—

ATTATGAATAGTTAGAAAGAGAGT	655	—

TTGTTGATATTAGAGAATGTGTTT	656	—

TTTATTGAGAGTTTGTTATTTGTG	657	—

AGTGTTAAGAAGTTGATTATTGAT	658	—

GAGAAATGATTGAATGTTGATAAT	659	—

GATAAGTATTAGTATGAGTGTAAT	660	—

TTTGATTTAAGAGTGTTGAATGTA	661	16

AAGTTAGTAAATAGAGTAGAAAGA	662	—

GTAAAGTATGAATATGTGAAATGT	663	—

TAATAAGTGTGTTGTGAATGTAAT	664	—

AAAGATTTAGAGTAGAAAGAGAAT	665	—

TTAGTTTGAGTTGAAATAGTAAAG	666	—

TAATAGTATGAGTAAGATTGAAAG	667	—

GAAGATTAGATTGATGTTAGTTAA	668	—

TAAAGAGAGAAGTTAGTAATAGAA	669	—

TAAGTATGAGAAATGATGTGTTAT	670	—

GAGTTTGTTTGTTAGTTATTGATA	671	—

AAGTAAAGAAATGTTAAGAGTAGT	672	—

ATGAGAATTGTTGTTGAAATGTAA	673	—

TTAGATTAGAGTAGTAGAAGAATA	674	—

TAGTGATGAAGAAGTTAGAAATTA	675	—

TAATGTAGTAATGTGATGATAAGT	676	—

TTGAGAAAGAATAAGTAGTGTAAA	677	—

TAATGAGTGAGATTATAGATTGTT	678	—

GTATAAGAAATGTGTGTTTGATTA	679	—

GTGAATGTGTTAATGAAGATATAT	680	—

GAAAGTTATTAGTAGTTAAAGATG	681	—

TAGAATTGTGTTTGATAAGTGATA	682	—

TGATTTAGATTGAGAGTTAAATGA	683	—

ATTATTGAGTTTGAATGTTGATAG	684	—

ATAGTAGTTATGTTTGATTTAGTG	685	—

ATAGAAGAAGAATAAAGTTAGAGA	686	—

GATGTTGAAAGTAATGAATTTGTA	687	—

GAGATTGATAGTAGAAATGATAAA	688	—

TGAGAGAATAAAGTATGAATTTGA	689	—

TATAAAGATGATGTGAATTAGTAG	690	—

TTATGTAAGAATGTTTGAGAGAAA	691	—

AGTAAATGATGAATGATATGATGA	692	—

GAAATTTGTGTTAAAGTTGAATGA	693	—

GATGAATGATTGTGTTTAAGTATA	694	—

GAAATAAGTGAGAGTTAATGAAAT	695	—

TGTTGAAATAGTTATTAGTTTGTG	696	—

TTTGAGAGTATATTGATATGAGAA	697	—

ATTGTGTGTAAAGTAAGATTTAAG	698	—

TATAGTTTGAAGTGTGATGTATTT	699	—

GTGAAGTTATAGTGTATAAAGAAT	700	—

GTATGTTGAATAGTAAATAGATTG	701	—

TTAGAAAGTGTGATTTGTGTATTT	702	—

TTTAGTAATATGTAAGAGATGTGA	703	—

AGTATGTATAGATGATGTTTGTTT	704	—

ATTTAAGTAAAGTGTAGAGATAAG	705	20

ATTTGTGTTGAATTGTAAAGTGAA	706	—

ATGTTATTAGATTGTGATGAATGA	707	—

TAGTAGTAGAATATGAAATTAGAG	708	—

TTTAATGAGAAGAGTTAGAGTATA	709	—

AAAGTTTAGTAGAGTGTATGTAAA	710	—

ATATATGATAGTAGAGTAGATTAG	711	—

TGAGAAGTTAATTGTATAGATTGA	712	—

TATAGAGATGTTATATGAAGTTGT	713	—

AAATTTGTTAAGTTGTTGTTGTTG	714	—

TTGTTGAAGATGAAAGTAGAATTA	715	—

AAGAGATAAGTAGTGTTTATGTTT	716	—

AATAAGAAGAAGTGAAAGATTGAT	717	—

TAAGTTAAAGTTGATGATTGATAG	718	—

ATATAAGATAAGAGTGTAAGTGAT	719	—

GTTAAATGTTGTTGTTTAAGTGAT	720	—

GAGTTAAGTTATTAGTTAAGAAGT	721	—

TATTAGAGTTTGAGAATAAGTAGT	722	21

TAATGTTGTTATGTGTTAGATGTT	723	—

GAAAGTTGATAGAATGTAATGTTT	724	—

TGATAGATGAATTGATTGATTAGT	725	—

ATGATAGAGTAAAGAATAAGTTGT	726	—

AGTAAGTGTTAGATAGTATTGAAT	727	27

ATGTAGATTAAAGTAGTGTATGTT	728	—

TTATTGATAATGAGAGAGTTAAAG	729	—

ATTTGTTATGATAAATGTGTAGTG	730	29

TTGAAGAAATAAGAGTAATAAGAG	731	—

TGTGTAATAAGTAGTAAGATTAGA	732	—

ATGAAAGTTAGAGTTTATGATAAG	733	37

ATTAGTTAAGAGAGTTTGTAGATT	734	—

TGTAGTATTGTATGATTAAAGTGT	735	—

AGTTGATAAAGAAGAAGAGTATAT	736	—

GTAATGAGATAAAGAGAGATAATT	737	—

TGTGTTGAAGATAAAGTTTATGAT	738	—

AAGAAGAGTAGTTAGAATTGATTA	739	—

GAATGAAGATGAAGTTTGTTAATA	740	—

AAATTGTTGAGATAAGATAGTGAT	741	—

TGATTGTTTAATGATGTGTGATTA	742	—

ATGAAGTATTGTTGAGTGATTTAA	743	—

GTGTAAATGTTTGAGATGTATATT	744	46

AATTGATGAGTTTAAAGAGTTGAT	745	—

TTTGTGTAATATGATTGAGAGTTT	746	—

GTAGTAGATGATTAAGAAGATAAA	747	—

TTTAATGTGAAATTTGTTGTGAGT	748	—

GTAAAGAATTAGATAAAGAGTGAT	749	—

AATAGTTAAGTTTAAGAGTTGTGT	750	—

GTGTGATGTTTATAGATTTGTTAT	751	—

GTATAGTGTGATTAGATTTGTAAA	752	49

GTTGTAAGAAAGATATGTAAGAAA	753	—

ATATTAGATTGTAAAGAGAGTGAA	754	—

GAGTGATATTGAAATTAGATTGTA	755	—

TAAGAAGTTAAAGAAGAGAGTTTA	756	—

GATGTTAGATAAAGTTTAAGTAGT	757	—

GTGATTGTATGAGAAATGTTAAAT	758	—

TGATTATTGTAAGAAAGATTGAGA	759	—

AAGAATTGTGTAAGTTTATGAGTA	760	—

TTGTATTTAGAAGATTTGTAGATG	761	—

TATATGTTTGTGTAAGAAGAAATG	762	—

GATAATGTGTGAATTTGTGAATAA	763	—

TTAGAAATGTGAGATTTAAGAGTT	764	—

AGTGTAGAATTTGTATTTAGTTGT	765	—

TAGTTAAGATAGAGTAAATGATAG	766	—

GAAGTGATATTGTAAATTGATAAG	767	—

GTAATTGTGTTAGATTTAAGAAGT	768	—

TGATATTTGTGAATTGATAGTATG	769	—

AAGTAAAGAGATATAGTTAAGTTG	770	—

ATTAGTTAAGTTATTTGTGAGTGA	771	—

AGATGAAGTAGTTTATGAATTAGA	772	—

TGAGTTAGTTAAGTGATAGTTAAA	773	—

TTATTGTAGATTTAGAGAAGATGA	774	—

TATTTGTGTTTGTTGATTAGATAG	775	—

GTATAATGTGTGTGAAAGTTATAA	776	—

TATATGTTGAGTATAAAGAGAGAA	777	—

TTAGTTAGTTTAAAGATTGTGAGT	778	—

TTTAGAATAAGTGATGTGATGAAA	779	—

AGAGTAATGTGTAAATAGTTAGAT	780	—

TGTGATAAAGAGAAATTAGTTGTT	781	—

GAATTTAGTGAATGTTTGAGATTA	782	—

TGTGATGTGTAAGTATATGAAATT	783	—

TTGTGAATGATTAATGAATAGAAG	784	51

AATGTTGTTTAGATTGAGAAAGTT	785	—

AGATTGTGTTAGTATTAGTATAAG	786	—

TTGATGTATTAGAAAGTTTATGTG	787	—

TATGATTGTGTGTTAGAGAATTTA	788	—

TAGTGTAGATATTTGATAGTTATG	789	52

AGTTTAATGTGTTTAGTTGTTATG	790	—

TGTGTAAAGTAGAAAGTAAAGATT	791	—

GTTATGATATAGTGAGTTGTTATT	792	53

TTTGATTGAATGTTAATAGTGTGT	793	—

AGAGTATTAGTAGTTATTGTAAGT	794	54

TAAGTAGAAAGAAGAAGATATTTG	795	—

AGAAAGAGAATTATGTAATGAAAG	796	—

TTAGATTTGTTAGTGTGATTTAAG	797	—

GATGATTAAGATATAGAGATAGTT	798	—

ATATTTGAGTGATTAAGAGTAATG	799	—

TGTATTGTGAGTTAAGTATAAGTT	800	—

AATTTAGTAGAAAGTGTTGTGTTT	801	—

GTTAGAAGATTAAGTTGAATAATG	802	—

TAAAGTATGTGAGATGATTTATGT	803	—

TGAAATGATTAAAGATGAAGATGA	804	—

TTATTAGATGTTGAGTGTTTGTTT	805	—

TAGTGTTTAAAGAGTAGTATATGA	806	—

AGTTATAAGTAAATGATGTTGATG	807	—

TTAAGAGAGAAATAAGTGTATTGT	808	—

GATATTGAAATGTGTAAATGATGA	809	—

ATGATGAATTAAGAAAGAAAGAGA	810	—

GAATAGTTTGATTTGTGTTTGTTA	811	—

AGTTGTTTAGATTTGATTTGTAAG	812	—

GTATGAGATTTGATATAAGATTAG	813	—

TTTATAGTGAGTATAGTGATGATT	814	—

TATATGTGAAGATATAAGTGTTTG	815	—

ATTGATAGATGATAGTAATTGAGT	816	—

TGATAGATGTGAAGAATTTGATTT	817	—

GAAGATATTGAAAGAATTTGATGT	818	55

GATGTTTAGTGTAGATATAGATTT	819	—

GAATATTGAGTTATAAGTAGTAGT	820	—

AGTGAGTAAGTAATAGAAAGATTT	821	—

GTAGAATAAGTAATTTGTGAGATA	822	—

GAGTTATTTGAGATTTAGATGTTT	823	—

GAAATGATGATTGAATTTAGAGAT	824	—

AAATAGTGTGAGAATAGTTAAGTA	825	—

ATGTGTTAAGTTGTAGAAGAATAA	826	—

ATAATGAGTTAATAGTGTAAGAAG	827	—

ATAAGAGATGTTTAAGTTAGAAAG	828	—

TGTTAGTGTTAGAAATATGAAAGA	829	—

TTTAGAAGATTGTTAGATAAGTTG	830	—

GTGTAATGTATAAGATAGTTAAGT	831	—

TATTAGAGAGAAATTGTAGAGATT	832	57

TAGTGAGATAAAGTAAAGTTTATG	833	—

TTGTGAAAGTTAAGTAAGTTAGTT	834	—

AAAGTGTAAGTTGAAGAATATTGA	835	—

GAATAGAGTGTTATTTGAAATAGA	836	—

TATAAGAGAGAGATAAGTAATAAG	837	—

TGAGTGAAATTGATAGAGTAAATT	838	—

GATGAATAAGTTTAAGTGAGAAAT	839	—

GTGTGATATGTTTATTGATTAAGT	840	—

TAAAGTGAGTGTAAATGATAATGA	841	—

GTAGAGTTTGATTTGAAAGAATAT	842	—

GAATATTGTTATGTTTGTTATGAG	843	—

GTGTAATAAGATGTATTGTTGTTT	844	—

TAAATTGATTGTGAGTTGAAGAAT	845	—

TGAGATAGTTATAGTTAAGTTTAG	846	—

AGTTTGTTAAGATTATGTAGAAAG	847	—

GAATGTGTAGAATAAGAGATTAAA	848	—

GTATTATGAAAGAAGTTGTTGTTT	849	—

GTGTTATAGAAGTTAAATGTTAAG	850	58

TTAAGAGTAGTGAATATGATAGTA	851	—

AATGTTATAAGATGAGAGTTTAGT	852	—

ATATAAGATTTGATGTAGTGTAGT	853	—

TATGTTTGTTGTTGTTAAGTTTGA	854	—

GATAGTTTAGTATAGAAGATAAAG	855	—

GTTGAATATAGAGATAGTAAATAG	856	—

AGAGAAGATTTAGTAAGAATGATA	857	—

TGAATGAGAAAGATATTGAGTATT	858	—

TGAAGATTATAGTAGTTGTATAGA	859	—

GATTAGTAGTATTGAAGATTATGT	860	—

TGAAATGTGTATTTGTATGTTTAG	861	59

ATTAAAGTTGATATGAAAGAAGTG	862	—

AATGTAGAGATTGTAGTGAATATT	863	62

TTATTTGTTGAGTGTAAATGTGAT	864	—

ATGTAATTGTGAATAATGTATGTG	865	63

GATTTGTATAGAGATTAGTAAGTA	866	—

AATATTGTTGTTTAGAGAAAGAAG	867	—

ATGATGATGTATTTGTAAAGAGTA	868	—

AATGTATTTGTGTGATTGTGTAAA	869	—

AGTGTTATGAAGAATAGTAAGAAT	870	—

GTTATGTAGAGATGAAAGAAATTA	871	65

GTTTGTATTAGATAAATGAGTTGT	872	—

TGATTTATGAGATTAAGAGAAAGA	873	—

TTTGTGTGTTATTGTAATTGAGAT	874	70

GATGTGTGATATGATTAAAGAAAT	875	—

AGATTATAGATTTGTAGAGAAAGT	876	—

GAAGAGTATGTAATAGTATTGTAT	877	—

TTTGTAATGTTGTTGAGTTTAAGA	878	—

AGTAAATAGTAGTATGAATAAGAG	879	—

GAATGTTGAATTGAAATATGAGTT	880	—

AGTAGTTAATTGATAGTAAGTTTG	881	—

AGTGTAAAGAAATGAATGAATAAG	882	—

TGTTAGATATTTGTGAAATGTGAA	883	—

TGTATGTTGAGTTTGAATTGTTAT	884	—

TGAGTGAATTAGTTATGTTGTTAT	885	—

GAAGAAAGAAATGAGAAAGATTAT	886	—

TTAAGTAAGTTGTGTTGATATTAG	887	—

ATGATGTGTTTGATTTGAATTGAA	888	72

AAGTAAGTGAAATTGTTGTTTGAA	889	—

ATGAAGTGTAAAGTTTGAAAGAAA	890	—

AGAGAGTAAGATAATTGTATAGTA	891	—

TTTATGAGATAGATGAAATAAGTG	892	—

AGAAATTAGTAGTAATGATTTGTG	893	—

GATTTGAGATTGAATGAGAATATA	894	—

GATTAGAAAGATGAATAAAGATGA	895	—

TAGATAGAAAGTATATGTTGTAGT	896	—

GAAGATAGTAAAGTAAAGTAAGTT	897	—

AAATGTGTGTTTAGTAGTTGTAAA	898	75

TTGTTGAAGTAAGAGATGAATAAA	899	—

TATTTGAGAGAAAGAAAGAGTTTA	900	—

TATTTAGTGATGAATTTGTGATGT	901	—

TTATAGTGATGATGATAAGTTGAT	902	—

TAAAGATAATTGTAGAAAGTAGTG	903	—

GTTTAGTATTGATATTGTGTGTAA	904	—

GTGTTGTGAATAAGATTGAAATAT	905	—

AAAGAAAGTATAAAGTGAGATAGA	906	—

TATTTGTAAGAAGTGTAGATATTG	907	—

TAGAAGATGAAATTGTGATTTGTT	908	—

ATAATAGTAAGTGAATGATGAGAT	909	—

AATGTGAATAAGATAAAGTGTGTA	910	—

ATTGAAGATAAAGATGTTGTTTAG	911	—

TGAAATAGAAGTGAGATTATAGTA	912	76

AGTTATTGTGAAAGAGTTTATGAT	913	—

AAATAGTAGTGATAGAGAAGATTT	914	—

AGTGTATGAAGTGTAATAAGATTA	915	—

TGATTAAGATTGTGTAGTGTTATA	916	—

AGTTTATGATATTTGTAGATGAGT	917	—

TATGTGTATGAAGATTATAGTTAG	918	78

GAAATTGTTGTATAGAGTGATATA	919	—

TAGAAATAGTTTAAGTATAGTGTG	920	—

TGATTTAGATGTTTATTGTGAGAA	921	—

AAGTTGATATTTGTTGTTAGATGA	922	—

TGATGTGATAATGAGAATAAAGAA	923	79

AAAGTTTAGTTTGTATTAGTAGAG	924	—

AGTTTGATGTGATAGTAAATAGAA	925	—

AAGTGTTATTGAATGTGATGTTAT	926	—

AAATTGAAGTGTGATAATGTTTGT	927	—

GTTTAGTGATTAAAGATAGATTAG	928	82

ATAAGTGTATAAGAGAAGTGTTAA	929	—

ATGAATTTGTTTGTGATGAAGTTA	930	—

AAAGAATTGAGAAATGAAAGTTAG	931	—

AGTGTAAGAGTATAAAGTATTTGA	932	—

GAATTAAGATTGTTATATGTGAGT	933	—

TATGAAAGTGTTGTTTAAGTAAGA	934	—

TAAAGTAAATGTTATGTGAGAGAA	935	—

AAAGATATTGATTGAGATAGAGTT	936	—

AAGTGATATGAATATGTGAGAAAT	937	—

AAATAGAGTTTGTTAATGTAAGTG	938	—

GATTTAGATGAGTTAAGAATTTAG	939	—

TTGTAAATGAGTGTGAATATTGTA	940	—

AGTAGTGTATTTGAGATAATAGAA	941	—

TGAGTTAAAGAGTTGTTGATATTT	942	—

AAAGAGTGTATTAGAAATAGTTTG	943	—

GTTTAGTTATTTGATGAGATAATG	944	—

AAGTGTAAATGAATAAAGAGTTGT	945	—

AATAAAGTGAGTAGAAGTGTAATT	946	—

TATTGAGTTTGTGTAAAGAAGATA	947	—

TTTATAGTTGTTGTGTTGAAAGTT	948	—

ATGAAATATGATTGTGTTTGTTGT	949	—

AAAGAGATGTAAAGTGAGTTATTA	950	—

TTGAAGAAAGTTAGATGATGAATT	951	—

ATGTTATTTGTTTAGTTTGTGTGA	952	—

AAATATGAATTTGAAGAGAAGTGA	953	—

GATTAGATATAGAATATTGAAGAG	954	—

TTAGAATAAGAGAAATGTATGTGT	955	—

TTTATGAAAGAGAAGTGTATTATG	956	—

GTAAGTATTAAGTGTGATTTAGTA	957	—

ATAAAGAGAAGTAAAGAGTAAAGT	958	—

ATTGTTAATTGAAGTGTATGAAAG	959	—

TATATAGTTGAGTTGAGTAAGATT	960	—

TAGATGAGATATATGAAAGATAGT	961	—

ATAAGAAGATGATTTGTGTAAATG	962	—

TTAGTAATAAGAAAGATGAAGAGA	963	—

GATTTGTGAGTAAAGTAAATAGAA	964	—

AAATAGATGTAGAATTTGTGTGTT	965	—

GAAATTAGTGTTTGTGTGTATTAT	966	—

ATTTGAGTATGATAGAAGATTGTT	967	—

ATAGAGTTGAAGTATGTAAAGTTT	968	—

TAATTTGTGAATGTTGTTATTGTG	969	—

TTAGTTTATGAGAGTGAGATTTAA	970	—

GTTGTTAGAGTGTTTATGAAATTT	971	—

TTTATTGTGATGTGAAATAAGAGA	972	—

GTAAGTAATATGATAGTGATTAAG	973	—

TGAGATGATGTATATGTAGTAATA	974	—

AATTGAGAAAGAGATAAATGATAG	975	85

TTTGAAGTGATGTTAGAATGTTTA	976	—

AGTTGTTGTGTAATTGTTAGTAAA	977	—

ATAGTGAGAAGTGATAAGATATTT	978	—

GTGTGATAAGTAATTGAGTTAAAT	979	—

TAGTTATTGTTTGTGAATTTGAGA	980	—

ATAGTTGAATAGTAATTTGAAGAG	981	—

ATGTTTGTGTTTGAATAGAGAATA	982	—

TGATAAAGATATGAGAGATTGTAA	983	—

TAAAGATGAGATGTTGTTAAAGTT	984	—

AAGTGAAATTTGTAAGAATTAGTG	985	—

GAAATGAGAGTTATTGATAGTTTA	986	—

TTTGTAAATGAGATATAGTGTTAG	987	—

GTTAATTGTGATATTTGATTAGTG	988	—

AGAGTGTTGATAAAGATGTTTATA	989	—

AATTGTGAGAAATTGATAAGAAGA	990	—

TTAAAGAGAATTGAGAAGAGAAAT	991	—

TTGTTAGAAGAATTGAATGTATGT	992	—

AGTTAAGATATGTGTGATGTTTAA	993	—

TGAGTTATGTTGTAATAGAAATTG	994	—

TTAGATAAGTTTAGAGATTGAGAA	995	—

ATGAGTAATAAGAGTATTTGAAGT	996	—

TGTTTAAGTGTAATGATTTGTTAG	997	—

TTGAAGAAGATTGTTATTGTTGAA	998	—

TATAGAAAGATTAAAGAGTGAATG	999	—

TAAATTGTTAGAAATTTGAGTGTG	1000	—

ATTGTTAGTGTGTTATTGATTATG	1001	—

GAGAATTATGTGTGAATATAGAAA	1002	—

TTGATTGATAAAGTAAAGAGTGTA	1003	—

GTGTGTAAATTGAATATGTTAATG	1004	—

AAAGTAAAGAAAGAAGTTTGAAAG	1005	—

TTTAGTTGAAGAATAGAAAGAAAG	1006	—

GTGTAATAAGAGTGAATAGTAATT	1007	—

TATTGAAATAAGAGAGATTTGTGA	1008	—

ATGAGAAAGAAGAAGTTAAGATTT	1009	—

AAGAGTGAGTATATTGTTAAAGAA	1010	—

TTTGTAAAGTGATGATGTAAGATA	1011	—

GATGTTATGTGATGAAATATGTAT	1012	—

GTAGAATAAAGTGTTAAAGTGTTA	1013	—

AAAGAGTATGTGTGTATGATATTT	1014	—

AAAGATAAGAGTTAGTAAATTGTG	1015	—

AAGAATTAGAGAATAAGTGTGATA	1016	—

GATAAGAAAGTGAAATGTAAATTG	1017	86

GATGAAAGATGTTTAAAGTTTGTT	1018	—

AGTGTAAGTAATAAGTTTGAGAAA	1019	—

GTTGAGAATTAGAATTTGATAAAG	1020	87

TTAAGAAATTTGTATGTGTTGTTG	1021	—

AGAAGATTTAGATGAAATGAGTTT	1022	—

TAAGTTTGAGATAAAGATGATATG	1023	—

TGAGATAGTTTGTAATATGTTTGT	1024	—

AGTTTGAAATTGTAAGTTTGATGA	1025	—

TAGAATTGATTAATGATGAGTAGT	1026	—

AGAGATTTGTAATAAGTATTGAAG	1027	—

ATAATGATGTAATGTAAGTAGTGT	1028	—

TGAAATTTGATGAGAGATATGTTA	1029	—

TGTGTAAAGTATAGTTTATGTTAG	1030	—

TGAATAAGTGAAATAGAATGAATG	1031	—

AAAGAAAGATTGTAATAAGTAGAG	1032	—

AATGAAATAGTGTTAAATGAGTGT	1033	89

GTAGATAAAGATGTGAATTATGAT	1034	—

GATAGTATATGTGTGTATTTGTTT	1035	—

ATGTTTGTAGAAATGTTTGAAGAT	1036	—

AAATTTGTAGAGAGAAATTTGTTG	1037	—

TAGAATAAGATTAGTAAGTGTAGA	1038	—

TGATTTAGAGAAATATGAGTAGAA	1039	—

AATAGAGTATGTTGTTTATGAGAA	1040	—

GATGATGAAGAGTTTATTGTAAAT	1041	—

AAGTAAAGAAGAAGAAATGTGTTA	1042	—

TTGAAGAATTAAGTGTTTAGTGTA	1043	—

AGAAAGAATGTTGATTTATGATGT	1044	—

GATTAAAGAGATGTTGATTGAAAT	1045	—

AATGATAATTGTTGAGAGAGTAAT	1046	—

GTTTGTTGAAAGTGTAAAGTATAT	1047	90

TGAGTTATATGAGAAAGTGTAATT	1048	—

TTGTGAGAAAGAAGTATATAGAAT	1049	—

GTAAGTTTAGAGTTATAGAGTTTA	1050	—

GATAGATAGATAAGTTAATTGAAG	1051	—

AGAGATGATTGTTTATGTATTATG	1052	—

AAAGTTAAGAAATTGTAGTGATAG	1053	—

TTTGATATTGTTTGTGAGTGTATA	1054	—

ATTTGTAGAAAGTTGTTATGAGTT	1055	—

GATTTGAGTAAGTTTATAGATGAA	1056	—

AAGATAAAGTGAGTTGATTTAGAT	1057	—

GATATTGTAAGATATGTTGTAAAG	1058	—

GTAAGAGTGTATTGTAAGTTAATT	1059	—

GTGTGATTAGTAATGAAGTATTTA	1060	91

GTAAGAAAGATTAAGTGTTAGTAA	1061	—

AGTAGAAAGTTGAAATTGATTATG	1062	92

TAAGAGAAGTTGAGTAATGTATTT	1063	—

GTTAAGAAATAGTAGATAAGTGAA	1064	—

TAAGTAAATTGAAAGTGTATAGTG	1065	—

AAGATGTATGTTTATTGTTGTGTA	1066	—

ATTTAGAATATAGTGAAGAGATAG	1067	—

GTTATGAAAGAGTATGTGTTAAAT	1068	93

TATTATGTGAAGAAGAATGATTAG	1069	—

TAATAAGTTGAAGAGAATTGTTGT	1070	—

TGATGTTTGATGTAATTGTTAAAG	1071	—

GTGAAAGATTTGAGTTTGTATAAT	1072	—

AGAGAATATAGATTGAGATTTGTT	1073	—

TTTGAGATGTGATGATAAAGTTAA	1074	—

GTTGTAAATTGTAGTAAAGAAGTA	1075	94

GTGTTATGATGTTGTTTGTATTAT	1076	—

ATTATTGTGTAGATGTATTAAGAG	1077	—

GTTAGAAAGATTTAGAAGTTAGTT	1078	—

TTGTGTATTAAGAGAGTGAAATAT	1079	—

GTTTAAGATAGAAAGAGTGATTTA	1080	—

AATGAGAAATAGATAGTTATTGTG	1081	—

TGAATTGAATAAGAATTTGTTGTG	1082	95

AATAAGATTGAATTAGTGAGTAAG	1083	—

AATGTTTGAGAGATTTAGTAAAGA	1084	—

AGTTTAGAATAGAAATGTGTTTGA	1085	—

TATAAGTAAGTGTTAAGATTTGAG	1086	—

GTAGTGAATAAGTTAGTGTTAATA	1087	—

AAGTGTGTTAAAGTAAATGTAGAT	1088	—

AGAGATGTTTATGTTGTGAATTAA	1089	—

AGTTGAATATTGATGATAAGAAGA	1090	—

TGAATGTGAGATGTTTAGAATAAT	1091	—

AATAATGATGTAAGTTTGAGTTTG	1092	—

AAAGAGTGAATAGAAATAAGAGAA	1093	—

AATAAAGTTATTGAGAGAGTTTAG	1094	—

AGTAGTGTTGTAGTTTAGTATATA	1095	—

GTAAGAATGTATTAGATATTTGTG	1096	—

GATAAATGTTTGATAAAGTAGTTG	1097	—

ATAGTATGTATGTGTGAAGATTTA	1098	—

ATGAATGTAGAGTGATTAGTTTAA	1099	—

GTAGTATTTAGTGATGTAAGAATA	1100	—

AGAATTGTATTGAAGAAGAATATG	1101	—

TTTATAGAATTGAGAGAAGTTAAG	1102	—

AAAGTAGTAGAGATTTGAGAATTA	1103	—

TTTAAAGAAAGTATTGTAAGAGTG	1104	—

AAATTGAGAAAGTGAATGAAGTTT	1105	—

AAGAAATAAGTATGATAGTAGTAG	1106	—

ATTTGAATTGTATTGTAGTTTGTG	1107	—

AAGAGAATAATGTAGAGATATAAG	1108	—

TGTGTAATAGTTGTTAATGAGTAA	1109	—

TATAGTTGTAGTTTAGATGAATGT	1110	—

ATTGTGTTAGAATGATGTTAATAG	1111	—

GTTTGTATAGTATTTGATTGATGT	1112	—

AGAGTAAAGTATGAGTTATGAATA	1113	—

GAAAGTTTAAGTGATGTATATTGT	1114	96

TTAAATGATAAAGAGTAGTGAAGT	1115	—

TTAAATGTGTGAGAAGATGAATAA	1116	—

ATTTGTATAAAGTGAAGAAGAGAA	1117	97

TGATTAGTATTTGTGAAGAGATTT	1118	—

TTTGAATGAAATTGATGATAGATG	1119	—

AGAGTAAGATTAAGAATAAGAAAG	1120	—

ATTGAATTGAGAAGTGAAGTAAAT	1121	—

TTTAGAGAAGTATTGTTTGAAAGA	1122	—

TAAAGTGAAAGATTTGAAATGATG	1123	—

GAAAGTTAGAGAAATGTAGAAATT	1124	—

GTGAATAATGAAGAAGTTATGTTA	1125	98

TTGTGAATAAAGTAGATGTGTTAT	1126	—

TTATATGATATGAGTTTGTGTTGA	1127	—

TTGATTTGTGTGAGTATTAGTTAT	1128	—

AAAGTGATTAAGTTAGTTTGAGAT	1129	—

TTGTATTTGTATAATGTTGAAGAG	1130	—

GTTTGAAATTAGTGTGAGAAATAT	1131	—

AATGTTGAGATTGATAATGTTGAA	1132	—

TAGTAGTAGTATTGTTGTAATAAG	1133	—

GTTGTAATTTGAGTGTTAGTTATT	1134	—

TGAATATGATAGTTAGTAATTGTG	1135	—

TGATAGTATGTTTGTGATTAAAGA	1136	—

GATGTATAAAGAGTATGTTATAAG	1137	—

AGTGAGATTTAGAAGATGTTATTA	1138	—

ATGAGAATTTGTTAAAGAGAAAGT	1139	—

AAAGAATTAGTATGATAGATGAGA	1140	99

TAGAGTTGTATAGTTTATAGTTGA	1141	—

GTAGAATGATTGTTTAGAAGATTT	1142	—

GTTTATGTTTGAGAAGAGTTATTT	1143	—

TAGAAGTTTGAAAGTTATTGATTG	1144	—

GATGAAGAGTATTTGTTATATGTA	1145	—

GATGAATATAGTAAGTATTGAGTA	1146	100

TAGTGATGAAATTTGAGATAGATA	1147	—

GAAAGAAATTGAAGAGTTTGATAT	1148	—

ATTTGAGTATTTGTGTATTGAATG	1149	—

ATGAGTTGAAATTTGAAGTATTGT	1150	—

TTAATAGTGAGAGAGTATATGTAA	1151	—

ATTAAGAGAGTGAGTAAATGTAAA	1152	—

AAGAATAGATGAGATTAGAAATAG	1153	—

AGTTTAAAGAGTTAGAATTGAAAG	1154	—

GTAAGATTTGTTGAATAAAGAAGA	1155	—

AGAGAAAGAAGTTAAAGTGATATT	1156	—

TAATAGAGAAGAGATGTATGAATA	1157	—

TTATTAGTGATAAGTGAAGTTTAG	1158	—

ATAATGTAAAGATGAGTTTATGAG	1159	—

TTGATTTGAGAGTTGATAAGATTT	1160	—

ATGATTATTGTGTGTAGAATTAGA	1161	—

TATAAAGATATAGTAGATGATGTG	1162	—

TTTAGTTGAGATGAAGTTATTAGA	1163	—

ATTGAATTGATATAGTGTAAAGTG	1164	—

GAAGAAAGATTATTGTATTGAGTT	1165	—

ATTGAGTGTAGTGATTTAGAAATA	1166	—

AATAAAGTGTTTAAGAGTAGAGTA	1167	—

GTAGAGATAATTGATGTGTAATTT	1168	—

[0398]

Patent Citations

Cited Patent	Filing date	Publication date	Applicant	Title
US4851331 *	May 16, 1986	Jul 25, 1989	Allied Corporation	Method and kit for polynucleotide assay including primer-dependant DNA polymerase
US4942124 *	Aug 4, 1988	Jul 17, 1990	President And Fellows Of Harvard College	Multiplex sequencing
US5391480 *	Dec 3, 1991	Feb 21, 1995	Collaborative Research, Inc.	Method for detecting a nucleotide at a specific location within a nucleic acid using exonuclease activity
US5654413 *	Jun 7, 1995	Aug 5, 1997	Spectragen, Inc.	Compositions for sorting polynucleotides
US5981176 *	Jun 17, 1992	Nov 9, 1999	City Of Hope	Method of detecting and discriminating between nucleic acid sequences
US6103463 *	May 23, 1994	Aug 15, 2000	The Public Health Research Institute Of The City Of New York, Inc.	Method of sorting a mixture of nucleic acid strands on a binary array
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* Cited by examiner

Referenced by

Citing Patent	Filing date	Publication date	Applicant	Title
US20060204971 *	Mar 11, 2005	Sep 14, 2006	Varde Shobha A	Oligonucleotides for multiplexed binding assays
US20080131875 *	Jun 7, 2006	Jun 5, 2008	Third Wave Technologies, Inc.	Multiplex assays
CN102010898A *	Jun 8, 2010	Apr 13, 2011	广州益善生物技术有限公司	EPHX1 (Microsomal epoxide hydrolase, mEH, EPHX1) gene detection specific primer and liquid phase chip
CN102021237A *	Jun 8, 2010	Apr 20, 2011	广州益善生物技术有限公司	Liquid phase chip and specificity primer for SNP detection of CYP4F2 and EPHX1 genes
CN102021237B	Jun 8, 2010	Aug 28, 2013	广州益善生物技术有限公司	Liquid phase chip and specificity primer for SNP detection of CYP4F2 and EPHX1 genes

* Cited by examiner

Classifications


U.S. Classification	435/6.18, 435/91.2, 536/23.1, 435/6.1
International Classification	C12Q1/68, C07H21/02, C12P19/34
Cooperative Classification	C07H21/02, C12Q1/6816
European Classification	C12Q1/68B2, C07H21/02

Legal Events

Date	Code	Event	Description
Feb 18, 2005	AS	Assignment	Owner name: TM BIOSCIENCE CORPORATION, CANADA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:JANECZKO, RICHARD ADAM;REEL/FRAME:015743/0295 Effective date: 20040920
Feb 16, 2007	AS	Assignment	Owner name: LAURUS MASTER FUND, LTD., NEW YORK Free format text: SECURITY AGREEMENT;ASSIGNOR:TM BIOSCIENCES CORPORATION;REEL/FRAME:018898/0348 Effective date: 20051122


*

A-A-B-B-C-A
A-A-D-D-C-D	(1 out of 1 paired bases are
	the same)


	221873 221896
	5′-Biotin-TCATGGCGACTGTCCAGCTTTGTG-3′


	221873 221896

	5′-Biotin-TCATGGCGACTGTCCAGCTTTGTG-3′

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